Prion protein antibodies for the treatment of alzheimer&#39;s disease

ABSTRACT

The invention relates to a ligand capable of binding PrP at a site within amino acid residues 131 to 153 of PrP, for use in treatment or prevention of impaired synaptic plasticity. The invention also relates to a ligand capable of binding PrP at a site within amino acid residues 131 to 153 of PrP, for use in treatment or prevention of toxicity of Aβ oligomers. The invention also relates to a ligand capable of binding PrP at a site within amino acid residues 131 to 153 of PrP, for use in treatment or prevention of Alzheimer&#39;s Disease. The invention also relates to methods of medical treatment.

FIELD OF THE INVENTION

The invention relates to applications of ligands binding at a specificregion of prion protein (PrP) for treating toxicity related to bindingof oligomeric Amyloid[beta] to PrP and medical conditions involvingsame.

BACKGROUND TO THE INVENTION

Prion protein is a well-characterised and studied protein. Major prionprotein (PrP) also known as CD230 (cluster of differentiation 230) is aprotein that in humans is encoded by the PRNP gene (PRioN Protein). Themajor prion protein is expressed in the brain and several other tissues.The human PRNP gene is located on the short (p) arm of chromosome 20between the end (terminus) of the arm and position 12, from base pairU.S. Pat. No. 4,615,068 to base pair U.S. Pat. No. 4,630,233. More than20 mutations in the PRNP gene have been identified in people withinherited prion diseases, which include Creutzfeldt-Jakob disease,Gerstmann-Sträussler-Scheinker syndrome and fatal familial insomnia.

Helix 1 is a well characterised alpha-helix within the structure of thePrP c form of the prion protein (cellular/common form of prion protein).Ligands that bind to helix 1 of the PrP^(C) are known, for exampleantibody CSM-18 as disclosed in WO2004/050120 and commercially availablefrom D-Gen Limited, UK.

Soluble non-fibrillar forms of A[beta] have been implicated in, andshown to correlate with, disease progression in animal models ofAlzheimer's disease (AD) and patients with AD⁹. Low nanomolarconcentrations of synthetic A[beta] are known to disrupt synapticplasticity in vivo and in vitro, but the conformation and size of theA[beta] species responsible remain unclear¹⁰⁻¹³. It has been reportedthat the prion protein (PrP^(C)) can act as a cellular receptor for apreparation of synthetic A[beta] referred to as A[beta]-deriveddiffusible ligands (ADDL) and that PrP^(C) is required for thedisruption of synaptic plasticity mediated by ADDL¹. Nanomolar affinityof binding has been reported^(3,4,14) and a single anti-PrP monoclonalantibody with an epitope around residues 95-105 blocked ADDL binding andtoxicityl. In further studies, constitutive knockout of PrP c expressionreversed several pathological phenotypes in a mouse model of AD² as didperipheral treatment of the same mouse model with an anti-PrPantibody¹⁵. Previous work, as disclosed in WO2008/13034, hasconcentrated on the ADDL binding site of PrP^(C), which is found atamino acid residues 95-105 of PrP.

Such a finding is of considerable importance given the extensiveinvestigation of targeting PrP^(C) for prion disease therapeutics¹⁶. Inparticular the abolition of neuronal PrP expression in the adult murinenervous system is without serious consequence^(17,18) and both smallmolecule¹⁶ and monoclonal antibody therapeutics⁶ have been extensivelystudied. Indeed therapeutic molecular interactions with PrP^(C) havebeen characterised and fully humanised anti-PrP monoclonal antibodieshave been produced for clinical studies in human prion disease^(8,19).

Using available materials, the ADDL binding site of PrP has beentargeted in the prior art.

The present invention seeks to overcome problem(s) associated with theprior art.

SUMMARY OF THE INVENTION

The focus in the art has been on the ADDL binding site of PrP. This isthe section of the PrP molecule which is believed to interact with theADDL. This has therefore been of maximal interest in the field. Thebinding site on PrP is characterised as being at amino acids 95-105 ofPrP. Blocking this site on PrP blocks the interaction with ADDL andameliorates binding.

In contrast to the prior art, the present inventors have targeted adifferent part of the PrP molecule. The present inventors have targetedthe 131-153 region of PrP. This includes the Helix 1 region of PrP.

It is surprising that targeting this region is effective. Firstly, thisregion is removed from the established ADDL binding site of PrP.Therefore it would not be expected to be effective in interfering withADDL binding.

Secondly, this region is at a very distinct spatial site compared to theADDL binding site. In fact this region is situated at the far oppositeside of the three-dimensional PrP molecule. Thus, the site is not onlyseparate in terms of the amino acid residues, but is also separate inthree dimensional space.

Therefore, it can be seen that the present invention provides analternative way of disrupting the ADDL-PrP binding which is different incharacter to the prior art methods. Moreover, it is itself surprisingthat it works, since it targets a completely different part of themolecule from the established ADDL binding site.

Thus, in one aspect the invention provides a ligand capable of binding,suitably stably binding, PrP at a site within amino acid residues 131 to153 of PrP, for use in treatment or prevention of impaired synapticplasticity.

Suitably the impaired synaptic plasticity is PrP-dependent impairedsynaptic plasticity.

In another aspect, the invention relates to a ligand capable of binding,suitably stably binding, PrP at a site within amino acid residues 131 to153 of PrP, for use in treatment or prevention of toxicity of Aβoligomers.

In another aspect, the invention relates to a ligand capable of binding,suitably stably binding, PrP at a site within amino acid residues 131 to153 of PrP, for use in treatment or prevention of Alzheimer's Disease.

Suitably said ligand is an antibody, scFv, or Fab, or other antigenbinding fragment thereof.

Suitably said ligand binds PrP at a site within amino acid residues 131to 153 of PrP with an affinity of 100 nM or less.

Suitably amino acid residues 131 to 153 of PrP have the sequenceGSAMSRPIIHFGSDYEDRYYREN.

In another aspect, the invention relates to use of a ligand as describedabove in the manufacture of a medicament for impaired synapticplasticity, or toxicity of Aβ oligomers, or Alzheimer's Disease.

In another aspect, the invention relates to a method of treatment ofimpaired synaptic plasticity, or toxicity of Aβ oligomers, orAlzheimer's Disease, said method comprising administering to a subject atherapeutically effective amount of a ligand as described above.

In another aspect, the invention relates to a method of prevention ofimpaired synaptic plasticity, or toxicity of Aβ oligomers, orAlzheimer's Disease, said method comprising administering to a subject atherapeutically effective amount of a ligand as described above.

In another aspect, the invention relates to use of a ligand as describedabove in the manufacture of a medicament for treatment of impairedsynaptic plasticity, or toxicity of Aβ oligomers, or Alzheimer'sDisease.

In another aspect, the invention relates to use of a ligand as describedabove in the manufacture of a medicament for prevention of impairedsynaptic plasticity, or toxicity of Aβ oligomers, or Alzheimer'sDisease.

In another aspect, the invention relates to a method of treatment ofimpaired synaptic plasticity, or toxicity of Aβ oligomers, orAlzheimer's Disease, said method comprising administering to a subject atherapeutically effective amount of a ligand as described above.

In another aspect, the invention relates to a method of prevention ofimpaired synaptic plasticity, or toxicity of Aβ oligomers, orAlzheimer's Disease, said method comprising administering to a subject atherapeutically effective amount of a ligand as described above.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1—Biophysical characterisation of A[beta] species present in thebADDL preparation

a, Size exclusion chromatography of two different batches of freshlyprepared ADDLs and bADDLs eluted in PBS showing the ratio of oligomeric(left) to monomeric (middle) A[beta], as well as a buffer peak (right).b, Velocity analytical ultra-centrifugation of freshly prepared bADDLsin Ham's F12 medium as detected by absorbance at 280 nm and representedby c(s), the sedimentation coefficient distribution. The monomer(calculated molecular mass 5,000-6,000) and the oligomer components(calculated molecular mass 90,000-400,000) have been coloured blue andred, respectively. c, Negatively stained transmission electronmicrograph of (i) bADDLs and (ii) ADDLs showing a mixture of globularand rod-like structures <100 nm in length. Scale bar 50 nm. d, SDS-PAGEof ADDL preparations analysed by (i) silver staining to estimate theamount of soluble A[beta] present in 3 different ADDL preparations and(ii) Western blot analysis using the N-terminal anti-A[beta] antibody,6E10, to identify low abundance SDS-stable A[beta] species. Twodifferent concentrations of each test sample (10 microM and 20 microM,for Silver stain and 1 microM and 2 microM for Western blot) wereexamined

FIG. 2—Size-exclusion chromatography (SEC) profile of both ADDLs andbADDLs

ADDLs and bADDLs (1 ml of 100 microM stock) were chromatographed on aSuperdex 75 (10/30HR) column and eluted with ACSF at a flow rate of 0.8ml/min. Peptides were detected by absorbance at 220 nm. The ADDL/bADDLpreparations produced two peaks, one eluting in the void volume after 8ml and the other eluting after 14 ml in a manner highly similar to thefreshly dissolved peptide (see FIG. 1 b). ADDLs and bADDLs eluted inHam's F12 medium produced a pattern highly similar to that shown aboveand in FIG. 1 b.

FIG. 3—Negatively stained transmission electron micrograph of ADDLs

a Vehicle alone. b ADDLs contain a mixture of globular structures andflexible protofibrils <100 nm in length. Scale bar used is 100 nm.

FIG. 4—PrPC is required for the inhibition of LTP by bADDLs andA[beta]-containing extracts of human brain fEPSPs were recorded from theCA1 region of the hippocampus in all cases. Insets show example fEPSPtraces before and 1 h. post theta-burst stimulation (TB, stimulusartefact was removed for clarity). a. Extracellular recordings from FVBmice show stable LTP measured up to one hour post-TBS (squares, 184±15%,n=7). Pre-treatment of the slices with bADDLS for 30 min prior to TBcaused a significant inhibition of LTP (circles, 109±10%, p<0.01, n=6).b, LTP was reliably induced in slices from PrP null mice treated withvehicle control (squares, 151±8%, n=6). Significantly, perfusion ofslices from PrP null mice with the same bADDL preparation used in a, didnot impair LTP (circles, 149±11%, n=5, P>0.05). c, IP/Western blotanalysis of brain extracts revealed the presence of abundant A[beta]monomer and SDS-stable dimer in a sample taken from an AD brain and thecomplete absence of A[beta] in an extract from a non-demented controlsubject (Ctrl). Estimates of A[beta]concentration indicate the presenceof 14 ng/ml and 3.2 ng/ml of monomer and SDS-stable dimer respectively.NS indicates non-specific immunoreactive bands detected whenTris-buffered saline (TBS) alone was immunoprecipitated. Molecularweight markers are on the left. M and D denote A[beta] monomer andSDS-stable dimer. d, Perfusion of slices from wild type FVB mice with ADbrain for 30 min prior to TB significantly impaired LTP (circles,116±9%, n=6) compared to slices perfused with control brain extract(squares, 153±8%, n=6, p<0.05). In contrast, treatment of slices fromPrP^(−/−) mice with AD brain extracts failed to alter LIP (triangles,164±10%, n=6, P>0.05). * refers to when perfusion of bADDLs/AD brainextract was started and arrow denotes application of TB (4 pulses @ 100Hz delivered ten times with an inter-train interval of 200 msec). Thenumbers on the EPSP samples correspond to time during the experimentfrom which they originate. All values are mean±SEM. Scale bar on insetsdenotes 1 mv, 5 msec.

FIG. 5—ADDLs and bADDLS potently inhibit hippocampal LTP

Treatment of C57BL/6J mice with 500 nM bADDLS ([triangle], 94±13%, n=5)and ADDLs ([circle] 115±7%, n=6) produced a significant depression ofLTP when compared to controls ([square] 154±6%, n=10, p<0.01). WithbADDLs tending to cause a greater depression than ADDLs. This trend mayrelate to the fact that bADDL preparations tended to have larger amountsof high molecular weight A[beta] species than did ADDLs (FIG. 1 b andFIG. 2). * denotes perfusion of ADDLs/bADDLs, arrow denotes theta-burststimulation. FIG. 6—bADDLs avidly bind PrP in manner that can be blockedby certain anti-PrP antibodies.

a, Dose response curves of ADDLs (filled in squares) and bADDLs (filledin circles) to plate-bound full-length PrP show both preparations bindto human PrP with apparent dissociation constants of approximately 100nM. Background wells coated with BSA show the interaction becomingnon-specific in the micromolar range (light green and red for ADDLs andbADDLs, respectively). b, At nominal A[beta] concentration of 100 nM asimilar level of bADDLs binds to huPrP₂₃₋₂₃₁ as to huPrP₉₁₋₂₃₁ withbackground levels binding to huPrP₁₁₉₋₂₃₁, suggests a single bindingsite at residues 91-119. c, Competition of binding of bADDLs tosurface-bound huPrP₂₃₋₂₃₁ by difference constructs of PrP shows thathuPrP₂₃₋₂₃₁ binds 100-fold tighter to bADDLs in solution thanhuPrP₉₁₋₂₃₁. d, Pre-incubation of surface-bound huPrP₂₃₋₂₃₁ with ICSM-35blocks the subsequent binding of bADDLs in a dose-dependent manner withan IC₅₀=10.4±1.7 microM. e, Screen of the ICSM panel of antibodies usinga high throughput DELFIA® assay shows that all antibodies that bind toPrP block bADDL binding, with those that recognise an epitope in theregion 95-105 (filled in square) inhibiting bADDL binding more than tothose that recognise an epitope within the region 131-153 (filled indiamond) or those that bind to the structured region (filled intriangle) or undefined epitopes (filled in circle). Surface boundhuPrP₂₃₋₂₃₁ was pre-incubated for 1 h with 10 nM antibodies and thenincubated for 1 h with or without 100 nM bADDLs prior to detection witheither Eu-N1 streptavidin or Eu-N1 anti-mouse antibody. f, i, Model ofthe PrP:ICSM-18 complex based on the published crystal structure andwith the antibody extension built in with PrP and the ICSM-18 epitope(yellow) highlighted; ii, Modelled structure of the PrP:A[beta]interaction with A[beta] spheroids (green), PrP (magenta), the ICSM-18epitope (yellow) and the unstructured ICSM-35 epitope (red) built in,highlighting the large distance between A[beta] binding site and theICSM-18 epitope. All graphs show Mean±standard deviation and are anaverage of at least three data points.

FIG. 7—bADDL binding to huPrP23-231 in the presence of differentconcentrations of EDTA

This demonstrates that the observed interaction between PrP and A[beta]is not caused by copper coordination unless the dissociation constant istighter than 10-22 M. Binding is shown relative to binding in theabsence of EDTA.

FIG. 8—When administered alone neither ICSM-35 nor ICSM-18 alters LTP a,TB conditioning stimulation in C57/B6 mice treated with 2[micro]g/mlICSM-35 (circle) induced LTP (157±17%, n=4) that was similar inmagnitude to LTP induced in controls (square) (153±7%, n=6). b,Similarly, TB conditioning stimulation in C57BL/6J mice treated with2[micro]g/ml ICSM-18 (triangle) induced LTP (150±13%, n=3) that was notsignificantly different from LTP induced in controls (square) (153±7%).c, Injection of ICSM-18 (circle) or an IgG1 isotype control antibody(triangle) i.c.v. (#, both 30[micro]g in 10[micro]l) 30 min beforeinjection of vehicle (*, 5[micro]l i.c.v.) did not significantly affectHFS-induced LTP in the anaesthetised rat (131±9% and 133±7%,respectively; p>0.05 compared with animals that received two (square)vehicle injections, 136±12%, n=3 per group, Mann-Whitney U test). Arrowdenotes the time of application of conditioning stimulation.

FIG. 9—Inhibition of LIP by ADDLs or A[beta]-containing AD brain extractis ameliorated by the anti-PrPc antibodies ICSM-35 and ICSM-18.

a, Conditioning stimulation in hippocampal slices from C57/B6J micetreated with vehicle solution induced a robust LTP (black squares,152±7%, n=6), whereas pre-treatment (30 min) with ADDLs significantlydepressed LTP (red circles, 115±5%, n=8, p<0.01). In contrast, perfusionof slices with the anti-PrP antibody, ICSM-35 (which recognises anepitope within residues 93-102) (2 [micro]g/ml), 20 min prior toapplication of ADDLs prevented the impairment of LTP caused by ADDLs(grey triangles, 151±9%, n=6, P>0.05). b, As in a above, but ICSM-18(which recognises an epitiope within residues 143-153 of PrP) was usedin place of ICSM-35. Like ICSM-35, ICSM-18 (grey triangles, 157±9%, n=6)completely ameliorated the inhibitory activity of ADDLs (p<0.01). #refers to perfusion of ICSM18/35, * refers to perfusion of ADDLs, arrowdenotes TB stimulation. Inset calibration: 1 mV, 5 ms, stimulus artifactwas removed for clarity. c, Synaptic field potentials were recorded invivo from the CA1 area of anaesthetised male Wistar rats. Invehicle-injected rats (#, first injection 10 [micro]l i.c.v.; *, secondinjection 5 [micro]l 30 min later) high-frequency stimulation (HFS)triggered persistent and stable LTP (black squares, 136±7% at 3 hpost-tetanus, n=5). In contrast, injection of 5 [micro]lA[beta]-containing brain extract 15 min before HFS, in animalspre-injected with an IgG1 isotype control antibody (30 [micro]g),significantly inhibited LTP (red circles, 106±4%, n=5, p<0.05).Importantly, injection of the anti-PrPc antibody, ICSM-18 30 min priorto injection of A[beta]-containing TBS AD brain TBS extract (15 minprior to HFS) prevented the inhibition of LTP (grey triangles, 136±5%,n=5). Calibration: 2 mV, 10 msec. # refers to injection of ICSM18, *refers to injection of TBS-extract of human derived A[beta], arrowdenotes HFS. Insets show representative electrophysiological traces atthe times indicated before (1,3,5) and after (2,4,6) conditioningstimulation.

FIG. 10—Characterisation of AD brain extract used for in vivoelectrophysiology.

IP/Western blot analysis of brain extracts revealed the presence ofabundant A[beta] monomer and SDS-stable dimer in the brain of an 80 yearold female diagnosed with Alzheimer's disease (AD) and the completeabsence of A[beta] in an immunodepleted (ID) sample of the same brain.NS indicates non-specific immunoreactive bands detected whenTris-buffered saline alone was immunoprecipitated (TBS). Molecularweight markers are on the left. M and D denote A[beta] monomer andSDS-stable dimer.-ve refers to TBS control.

FIG. 11 and FIG. 12 each show annotated antibody sequences. Note: yellow(boxed) residues are CDRs; grey shaded (unboxed, bold) is the start ofthe constant region; regions which are neither leader sequence, constantregion nor CDR are defined as framework sequence.

FIG. 13—The mode of antibody-induced inhibition of ADDL binding tohuPrP₂₃₋₂₃1. a, Relative binding of monomeric (▴) and oligomeric (▪)Aβ₁₋₄₂ to surface-bound huPrP₂₃₋₂₃₁ detected using 6E10 and DELFIA®Eu-N1 anti-mouse antibodies. b, binding of ICSM-18 (▪) and ICSM-35 ()to surface-bound huPrP₂₃₋₂₃₁ detected using 6E10 and DELFIA® Eu-N1anti-mouse antibodies. c, Inhibition of bADDL binding to surface-boundhuPrP₂₃₋₂₃₁ by ICSM-18 (▪) and ICSM-35 () and detected using DELFIA®Eu-N1 streptavidin. Error bars show standard deviations and are theaverage of at least three replicates.

FIG. 14 shows a graph. Synaptic field potentials were recorded in vivofrom the CA1 area of anaesthetised male Wistar rats. In vehicle-injectedrats (#, first injection 10 [micro]l i.c.v.; *, second injection 5[micro]l 30 min later) high-frequency stimulation (HFS) triggeredpersistent and stable LTP (black squares, at 3 h post-tetanus). Incontrast, injection of 5 [microl]l A[beta]-containing brain extract 15min before HFS, in animals pre-injected with an IgG4 isotype controlantibody (30 [micro]g), significantly inhibited LTP (pink circles) asdid A[beta]-containing brain extract alone (red circles). Importantly,injection of the anti-PrPc antibody, PRN 100 30 min prior to toinjection of A[beta]-containing TBS AD brain TBS extract (15 min priorto HFS) prevented the inhibition of LTP (grey triangles). #refers toinjection of PRN100 or control IgG4, * refers to injection ofTBS-extract of human derived A[beta], arrow denotes HFS.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The term ‘comprises’ (comprise, comprising) should be understood to haveits normal meaning in the art, i.e. that the stated feature or group offeatures is included, but that the term does not exclude any otherstated feature or group of febtures from also being present.

Prion Protein (PrP)

When particular amino acid residues are referred to using numericaddresses, the numbering is taken using human prion protein amino acidsequence as the reference sequence. This is to be used as is wellunderstood in the art to locate the residue of interest. This is notalways a strict counting exercise—attention must be paid to the context.For example, if the protein of interest such as human PrP is of aslightly different length, then location of the correct residue in thehuman sequence correseponding to (for example) residue 131 may requirethe sequences to be aligned and the equivalent or corresponding residuepicked, rather than simply taking the 131st residue of the sequence ofinterest. This is well within the ambit of the skilled reader.

The reference sequence is suitably amino acid sequence from residue 23to 231 of human PrP:

23KKRPKPGG WNTGGSRYPG QGSPGGNRYP PQGGGGWGQPHGGGWGQPHG GGWGQPHGGG WGQPHGGGWG QGGGTHSQWNKPSKPKTNMK HMAGAAAAGA VVGGLGGYML GSAMSRPIIH FGSDYEDRYY RENM HRYPNQ VYYRPMDEYS NQNNFVHDCV NITIKQHTVT TTTKGENFTE TDVKMMERVV EQMCITQYER ESQAYYQRGS 231S

Target sequence is marked with underlining/bold. The S1-H1 loop areahighlighted by underlining. Helix-1 of PrP is highlighted in bold. Thistarget sequence is well conserved between species.

The prion protein (PrP) has already been well characterised. The prionprotein sequence may be derived from the human PRNP gene. More suitably,the protein sequence is the sequence between amino acid residues 23 and231 as shown above.

Suitably the prion protein to which the ligand according to theinvention binds to is PrP^(C) (cellular/common form prion protein)conformation.

Target Region of PrP

The target region of PrP of maximal interest is the sheet 1-helix 1 loop(the S1H1 loop). This target region suitably comprises helix 1 plus asegment of unstructured sequence. This is sometimes referred to as helix1 plus the loop between beta strand 1 and alpha helix 1.

More suitably the target region comprises helix 1 of PrP. Helix 1 issometimes defined as including amino acids 143 to 156 of PrP^(C). Moresuitably the target region includes aa 143 to 153 or helix 1.

More suitably the target region comprises amino acids 131-153 of PrP.

More suitably the target region may comprise aa 131-150 of PrP, moresuitably aa 142-153 of PrP, most suitably aa 136-143 of PrP.

Suitably PrP is PrP.

PrP may be from any mammalian species such as cow, sheep, mouse,hamster, human or other mammal. Suitably PrP is livestock or human PrP.

Suitably PrP is human PrP.

Suitably the target region excludes the ADDL binding site aa 95-105 ofPrP, which has been described in the art as acknowledged herein. Theinvention is different from this because a target site separate anddistinct from the ADDL binding site is targeted herein. Suitably thetarget site does not comprise aa 95-105 of PrP. Suitably the target sitedoes not overlap with aa 95 to 105 of PrP.

Mutation

Mutating has it normal meaning in the art and may refer to thesubstitution or truncation or deletion of the residue, motif or domainreferred to. Mutation may be effected at the polypeptide level e.g. bysynthesis of a polypeptide having the mutated sequence, or may beeffected at the nucleotide level e.g. by making a nucleic acid encodingthe mutated sequence, which nucleic acid may be subsequently translatedto produce the mutated polypeptide. Where no amino acid is specified asthe replacement amino acid for a given mutation site, suitably alanine(A) is used.

Conservative substitutions may be made, for example according to theTable below. Amino acids in the same block in the second column andpreferably in the same line in the third column may be substituted foreach other:

ALIPHATIC Non-polar G A P M I L V Polar - uncharged C S T N QPolar - charged D E K R AROMATIC H F W Y

Fragment

A fragment is suitably at least 10 amino acids in length, suitably atleast 25 amino acids, suitably at least 50 amino acids, suitably atleast 100 amino acids, suitably at least 200 amino acids, suitably themajority of the polypeptide of interest. Suitably a fragment comprises awhole motif or a whole domain of the polypeptide of interest.

Sequence Homology/Identity

Although sequence homology can also be considered in terms of functionalsimilarity (i.e., amino acid residues having similar chemicalproperties/functions), in the context of the present document it ispreferred to express homology in terms of sequence identity.

Sequence comparisons can be conducted by eye or, more usually, with theaid of readily available sequence comparison programs. These publiclyand commercially available computer programs can calculate percenthomology (such as percent identity) between two or more sequences.

Percent identity may be calculated over contiguous sequences, i.e., onesequence is aligned with the other sequence and each amino acid in onesequence is directly compared with the corresponding amino acid in theother sequence, one residue at a time. This is called an “ungapped”alignment. Typically, such ungapped alignments are performed only over arelatively short number of residues (for example less than 50 contiguousamino acids).

Although this is a very simple and consistent method, it fails to takeinto consideration that, for example in an otherwise identical pair ofsequences, one insertion or deletion will cause the following amino acidresidues to be put out of alignment, thus potentially resulting in alarge reduction in percent homology (percent identity) when a globalalignment (an alignment across the whole sequence) is performed.Consequently, most sequence comparison methods are designed to produceoptimal alignments that take into consideration possible insertions anddeletions without penalising unduly the overall homology (identity)score. This is achieved by inserting “gaps” in the sequence alignment totry to maximise local homology/identity.

These more complex methods assign “gap penalties” to each gap thatoccurs in the alignment so that, for the same number of identical aminoacids, a sequence alignment with as few gaps as possible—reflectinghigher relatedness between the two compared sequences—will achieve ahigher score than one with many gaps. “Affine gap costs” are typicallyused that charge a relatively high cost for the existence of a gap and asmaller penalty for each subsequent residue in the gap. This is the mostcommonly used gap scoring system. High gap penalties will of courseproduce optimised alignments with fewer gaps. Most alignment programsallow the gap penalties to be modified. However, it is preferred to usethe default values when using such software for sequence comparisons.For example when using the GCG Wisconsin Bestfit package (see below) thedefault gap penalty for amino acid sequences is −12 for a gap and −4 foreach extension.

Calculation of maximum percent homology therefore firstly requires theproduction of an optimal alignment, taking into consideration gappenalties. A suitable computer program for carrying out such analignment is the GCG Wisconsin Bestfit package (University of Wisconsin,U.S.A; Devereux et al., 1984, Nucleic Acids Research 12:387). Examplesof other software than can perform sequence comparisons include, but arenot limited to, the BLAST package, FASTA (Altschul et al., 1990, J. Mol.Biol. 215:403-410) and the GENEWORKS suite of comparison tools.

Although the final percent homology can be measured in terms ofidentity, the alignment process itself is typically not based on anall-or-nothing pair comparison. Instead, a scaled similarity scorematrix is generally used that assigns scores to each pairwise comparisonbased on chemical similarity or evolutionary distance. An example ofsuch a matrix commonly used is the BLOSUM62 matrix—the default matrixfor the BLAST suite of programs. GCG Wisconsin programs generally useeither the public default values or a custom symbol comparison table ifsupplied. It is preferred to use the public default values for the GCGpackage, or in the case of other software, the default matrix, such asBLOSUM62. Once the software has produced an optimal alignment, it ispossible to calculate percent homology, preferably percent sequenceidentity. The software typically does this as part of the sequencecomparison and generates a numerical result.

In the context of the present document, a homologous amino acid sequenceis taken to include an amino acid sequence which is at least 15, 20, 25,30, 40, 50, 60, 70, 80 or 90% identical, preferably at least 95 or 98%identical at the amino acid level. Suitably said comparison is made overat least 50 or 100, preferably 200, 300, 400 or 500 amino acids with anyone of the relevant polypeptide sequences disclosed herein, mostsuitably across the full length of the polypeptide of interest.Suitably, homology should be considered with respect to one or more ofthose regions of the sequence known to be essential for protein functionrather than non-essential neighbouring sequences. This is especiallyimportant when considering homologous sequences from distantly relatedorganisms.

The same considerations apply to nucleic acid nucleotide sequences.

The term ‘derived from’ has its normal meaning in the art, wherein asubstance is considered to be ‘derived from’ a first substance when partof the substance has been created or constructed through a chain ofevents which incorporates all or part of the first substance into thesubstance in question. Naturally the two substances are likely to differe.g. through mutation, addition or deletion or similar modification, butif the substance in question has inherited features from the firstsubstance then it is derived from it. In particular, when used inconnection with biopolymers such as polynucleotide(s) or polypeptide(s),a substance is considered to be derived from a first substance when itpossesses sufficient sequence identity to be recognised as related tothe first substance. In this context, if a substance is derived from afirst substance, then said substance preferably has at least 10contiguous residues which possess at least 25% identity with the firstsubstance, preferably 30% identity, preferably 40% identity, preferably50% identity, preferably 60% identity, preferably 70% identity,preferably 80% identity, preferably 90% identity, preferably 95%identity, preferably 96% identity, preferably 97% identity, preferably98% identity, preferably 99% identity or even more. Preferably saidsubstance has at least 15 contiguous residues with said identity,preferably at least 20 residues, preferably at least 30 residues,preferably at least 50 residues, preferably at least 100 residues,preferably at least 200 residues, or even more. For multimeric entities,the term may be applied to the complex and/or to individual componentsas will be apparent from the context. Generally it will be enough if oneof the subunits is derived from the given entity.

Abeta

A[beta] oligomerisation can have deleterious effects. Abetaoligomerisation is implicated in various medical conditions, and theuses and methods described advantageously counteract such conditions.

Thus the invention relates to the treatment and/or prevention of medicalcondition(s) linked to A[beta] oligomerisation. Examples of theseinclude Lewy body dementia and inclusion body myositis. Abetaoligomerisation can take the form. of plaque formation. An importantmedical condition involving plaque formation is Alzheimer's Disease(AD).

A[beta]-derived diffusible ligands (ADDL) are synthetic solubleoligomeric non-fibrillar forms of A[beta] (monomer) which can be used tostudy A[beta] oligomerisation. ADDL inhibits long term potentiation(LTP). LTP is connected with synaptic plasticity. Thus the inventionfinds application in medical conditions which manifest abnormal synapticplasticity such as impaired synaptic plasticity. Synaptic plasticity isthe ability of the synapse between two neurons to change in strength inresponse to either use or disuse of transmission over synaptic pathways.Medical conditions that manifest abnormal synaptic plasticity includecognitive maladies such as Alzheimer's Disease.

Suitably the ligand according to the invention is for use in preventingor treating medical conditions linked to A[beta] oligomerisation, moresuitably the medical condition is Alzheimer's Disease (AD).

AD is a heterogeneous disease and may be considered aclinicopathological syndrome. As such, a number of studies have reporteddeleterious effects of A[beta] that do not require PrP expression³⁻⁵. Inthis context it is important to recognise that a range of differentA[beta] preparations have been used and that this may in part explainsome of the conflicting reports. Moreover, discrepancies between animal,tissue, cellular and biochemical AD models with respect to a possiblerole for PrPC are to be anticipated. Even given that PrP c is animportant receptor for toxic A[beta] species, it would not be expectedthat PrP c ablation would rescue all aspects of pathology in each model.Further, given that the concentration of the active species causingdisease in the studies is not always known and may differ betweenstudies by different groups some effects may be due to higher A[beta]concentrations which could elicit non-specific toxic effects. Withoutwishing to be bound by theory, suitably when the invention is applied toAD or Abeta toxicity, the AD or Abeta toxicity is PrP-mediated AD orAbeta toxicity.

Ligands

PrP binding ligands of the invention are suitably ligands binding thetarget region of PrP as described, suitably binding the target region ofPrP c as described.

The ligand may be a single entity or it may be a combination ofentities. Suitably it is a single entity.

The ligand may be an organic compound or other chemical, whether naturalor artificial. The ligand may be an amino acid molecule, a polypeptide,or a chemical derivative thereof, or a combination thereof. The ligandmay be designed or obtained from a library of compounds, which maycomprise peptides, as well as other compounds, such as small organicmolecules. By way of example, the ligand may be a natural substance, abiological macromolecule, or an extract made from biological materialssuch as bacteria, fungi, or animal (particularly mammalian) cells ortissues, an organic or an inorganic molecule, a synthetic agent, asemi-synthetic agent, a structural or functional mimetic, a peptide, apeptidomimetic, a derivatised agent, a peptide cleaved from a wholeprotein, or a peptide synthesised synthetically (such as, by way ofexample, either using a peptide synthesiser or by recombinant techniquesor combinations thereof, a recombinant ligand, an antibody, a natural ora non-natural ligand, a fusion protein or equivalent thereof andmutants, derivatives or combinations thereof).

Ligands that bind to the target region of PrP are known in the art.

The ligand may be a protein. One example of such a ligand isTetraspanin-7.

Suitably the ligand may be an antibody, or an antibody derivative suchas a scFv, or Fab. The term “antibody” as used herein is also intendedto encompass antibodies, digestion fragments, portions or variantsthereof, including antibody mimetics, or comprising portions ofantibodies that mimic the structure and/or function of an antibody orfragment or portion thereof, including single chain antibodies andfragments thereof. The important function to be preserved in each caseis recognition of (i.e. effective binding to) the target region of PrP.Typically this recognition/binding function is mediated by thecomplementarity determining regions (CDRs) of the antibody. Thussuitably the ligand of the invention or fragment thereof comprises CDRsrecognising the target region of PrP. Functional fragments includeantigen binding fragments that bind to the target region of PrP. Forexample, antibody fragments capable of binding including, but notlimited to Fab (e.g., by papain digestion), facb (e.g., by plasmindigestion), pFc′ (e.g., by pepsin or plasmin digestion), Fd (e.g., bypepsin digestion, partial reduction and reaggregation), Fv or scFv(e.g., by molecular biology techniques) fragments, are encompassed bythe present invention. Antibody fragments are also intended to include,e.g., domain deleted antibodies, diabodies, linear antibodies,single-chain antibody molecules, and multispecific antibodies formedfrom antibody fragments. Examples of antibodies that bind the targetregion, of PrP are known in the art. Examples include ICSM17 (for aaresidues 131-150; available from D-Gen Ltd, UK), ICSM18 (for aa residues142-153; available from D-Gen Ltd, UK), ICSM 30 (for aa residues136-143; available from D-Gen Ltd, UK) and ICSM 31 (for aa residues136-143; available from D-Gen Ltd, UK).

ICSM 32 (131-150; available from D-Gen Ltd, UK) also this spans thetarget region.

Antibody Sha31 (Medicorp Inc.; Alier et al 2011 J. Neurosci. vol 31pages 16292-16297) binds to residues 145-152 of PrP and therefore alsobinds within the target region. Sha31 antibody is available from BertinPharma (subsidiary of Bertin Technologies), France, as product numberA03213.

Other known helix-1 binding antibodies (for example, 6H4 (e.g. fromPrionics AG, Wagistrasse 27a, CH-8952 Schlieren-Zurich, Switzerland);E12/2 (e.g. as published in Cernilec et al 2007 Immunol Lett. October31; vol 113(1) pages 29-39. Epub 2007 Aug. 20.); D18 e.g. as publishedin Peretz et al 2001 Nature vol 412, pages 739-743) may find applicationin the invention.

A preferred example of such an antibody is ICSM-18 as used in theExamples below. More suitably such an antibody is a humanised version ofICSM-18 such as PRN 100.

The antibodies may be in any suitable form known in the art fortherapeutic use. The antibodies may be whole or fragments thereof.

In more detail, Suitably the ligand may be an antibody, or an antibodyderivative such as a scFv, or Fab. The term “antibody” as used herein isintended to encompass “immunoglobulins” and derivatives thereof.Immunoglobulins comprise various broad classes of polypeptides that canbe distinguished biochemically. In many examples, immunoglobulinsconsist of combination heavy chains and light chains. All immunoglobulinclasses including IgM, IgA, IgD, IgE, IgG and IgY and where appropriate,their subclasses, are clearly within the scope of the present invention.The following discussion will generally be directed to the IgG class ofimmunoglobulin molecules. With regard to IgG, a standard immunoglobulinmolecule comprises two identical light chain polypeptides of molecularweight approximately 25 kDa, and two identical heavy chain polypeptidesof approximate molecular weight 50 kDa. The resulting molecule, which isconventionally referred to as an IgG “monomer” consists of identicalhalves and the four chains that are typically joined by disulfide bondsin a “Y” configuration wherein the light chains adjoin the heavy chainsstarting at the mouth of the “Y” and continuing through the variableregion or domain. It is well recognised by those skilled in the art thatimmunoglobulins can be characterised in terms of variable and constantdomains. In this regard, it will be appreciated that the variabledomains of both the light (VL) and heavy (VH) chain portions determineantigen recognition and specificity. Conversely, the constant domains ofthe light chain (CL) and the heavy chain (normally consisting of CH1,CH2 or CH3 domains) confer important biological properties such assecretion, transplacental mobility, Fc receptor binding, complementbinding, and the like. As indicated above, the variable region allowsthe antibody to selectively recognize and specifically bind epitopes onantigens. That is, the VL domain and VH domain of an antibody combine toform the variable region that defines a three dimensional antigenbinding site, this site is also called the “antigen receptor”. Thisantibody structure forms the antigen binding site or antigen receptorpresent at the end of each arm of the Y. More specifically, the antigenbinding site is defined by three complementarity determining regions(CDRs) on each of the VH and VL chains. Thus within the amino acidsequence of a variable domain of an antibody there are three CDRs (knownas CDR1, CDR2 and CDR3). Since most sequence variation associated withimmunoglobulins is found in the CDRs, these regions are sometimesreferred to as “hypervariable regions”, among these CDRs, CDR3 shows thegreatest variability. Since the antigen binding sites are typicallycomposed of two variable domains (on two different polypeptide chainsbeing the heavy and light chain), there are six CDRs for each antigenreceptor that can collectively come into contact with the antigen. Thusa single IgG molecule has two antigen receptors, and therefore consistsof twelve CDRs. CDRs can also be referred to as “idiotypes”. In someinstances, for example certain immunoglobulin molecules derived fromcamelid species or engineered molecules based on camelidimmunoglobulins, a complete immunoglobulin molecule may consist of heavychains only, with no light chains. See, e.g., Hamers Casterman et al,Nature 363:446 448 (1993).

The ligand may be an antigen binding molecule, such as in oneembodiment, an antigen binding molecule of the invention comprises atleast one heavy or light chain CDR of an antibody molecule. In anotherembodiment, an antigen binding molecule of the invention comprises atleast two CDRs from one or more antibody molecules. In anotherembodiment, an antigen binding molecule of the invention comprises atleast three CDRs from one or more antibody molecules, in anotherembodiment, an antigen binding molecule of the invention comprises atleast four CDRs from one or more antibody molecules. In anotherembodiment, an antigen binding molecule of the invention comprises atleast five CDRs from one or more antibody molecules. In anotherembodiment, an antigen binding molecule of the invention comprises atleast six CDRs from one or more antibody molecules. Antibodies orimmunospecific fragments thereof for use in the methods of the inventioninclude, but are not limited to, polyclonal, monoclonal, multispecific,human, humanized, primatized, or chimeric antibodies, single chainantibodies, epitope-binding fragments, e.g., Fab, Fab′ and F(ab′)2, Fd,Fvs, single-chain Fvs (scFv), single-chain antibodies, disulfide-linkedFvs (sdFv), fragments comprising either a VL or VH domain, fragmentsproduced by a Fab expression library, and anti-idiotypic (anti-Id)antibodies (including, e.g., anti-Id antibodies to binding moleculesdisclosed herein). ScFv molecules are known in the art and are producedusing recombinant DNA technology as described in the Winter patent(ref). Immunoglobulin or antibody molecules of the invention can be ofany type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1,IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.The term “antibody” as used herein is also intended to encompassantibodies, digestion fragments, specified portions and variantsthereof, including antibody mimetics or comprising portions ofantibodies that mimic the structure and/or function of an antibody orspecified fragment or portion thereof, including single chain antibodiesand fragments thereof; each containing at least one CDR. See Qiu et al.,Nature Biotechnology 25:921-929 (2007). Functional fragments includeantigen binding fragments that bind to a PrPC antigen. For example,antibody fragments capable of binding to PrP or a portion thereof,including, but not limited to Fab (e.g., by papain digestion), facb(e.g., by plasmin digestion), pFc′ (e.g., by pepsin or plasmindigestion), Fd (e.g., by pepsin digestion, partial reduction andreaggregation), Fv or scFv (e.g., by molecular biology techniques)fragments, are encompassed by the present current invention. Antibodyfragments are also intended to include for example, domain deletedantibodies, linear antibodies, single-chain antibody molecules,multispecific antibodies formed from antibody fragments and diabodies.Diabodies are formed by the creation of scFvs with linker peptides thatare too short for the two variable regions to fold together (about fiveamino acids), forcing scFvs to dimerize. Diabodies have been shown tohave dissociation constants up to 40-fold lower than correspondingscFvs, meaning that they have a much higher affinity to their target.Consequently, drugs based on diabodies could in principle be used atmuch lower doses than other therapeutic antibodies. Modified versions ofeach of these categories of recombinant antibody fragments andcombinations thereof will be discernible to the skilled person and arewithin the scope of the current invention. Antibody fragments, includingsingle-chain antibodies, may comprise the variable region(s) alone or incombination with the entirety or a portion of the following: hingeregion, CH1, CH2, and CH3 domains. Also included in the invention areantigen-binding fragments also comprising any combination of variableregion(s) with a hinge region, CH1, CH2, and CH3 domains. Antibodies orimmunospecific fragments thereof for use in the therapeutic methodsdisclosed herein may be from any animal origin including birds andmammals. Preferably, the antibodies are human, murine, donkey, rabbit,goat, guinea pig, camel, llama, horse, or chicken antibodies. In anotherembodiment, the variable region may be chondrichthoid in origin (e.g.,from sharks). As used herein, “human” antibodies include antibodieshaving the amino acid sequence of a human immunoglobulin and includeantibodies isolated from human immunoglobulin libraries or from animalstransgenic for one or more human immunoglobulins and that do not expressendogenous immunoglobulins, as described below and, for example in, U.S.Pat. No. 5,939,598 by Kucherlapati et al. In certain PrPC antibodies orimmunospecific fragments thereof for use in the treatment methodsdisclosed herein, the heavy chain portions of one polypeptide chain of amultimer are identical to those on a second polypeptide chain of themultimer. Alternatively, heavy chain portion-containing monomers for usein the methods of the invention are not identical. For example, eachmonomer may comprise a different target binding site, forming, forexample, a bispecific antibody.

The antibodies may be humanised. Humanisation of antibodies is known inthe art and can be easily accomplished by the skilled worker. Forexample, ICSM18 may be advantageously humanised with reference to thesequences encoding the CDRs. Suitably the antibody comprises at leastthe CDRs of one or more antibodies described herein.

In this regard, the following sequence corresponds to ICSM18VH:

ICSM18VH ATGGAATGGAGCTGGGTTTTCCTOTTCCTCCTGTCAGGAACTGCAGGTGTCCTCTCTGAGGTCCAGCTACAACAGTCTGGACCTGAGCTGGTGAAGCCTGGGTOTTCAGTGAAgATATCCTGCAAGGCATCTAGAAACACATTCACTGACTATAACTTGGACTGGGTGAAGCAGAGCCATGGAAAGACACTTGAGTGGATTGGAAATGTTTATCCTAACAATGGTGTTACTGGCTACAACCAgAAgTTCAGGGGTAAGGCCACACTGACTGTAgACAAGTCCTCCAGCACAGCCTACATGGAGCTCCACAGCCTGACATCTGAGGACTCTGCAGTOTATTACTGTGCCCTTTATTACTACgATgTCTCTTACTGGGGCCAAGGGACTCTGGTCACTGTCT CTGCA

The following sequence corresponds to ICSM181c:

ICSM181c ATGGATTTACAGGTGCAGATTATCAGCTTCCTGCTAATCAGTGCCTCAGTCATAATATCCAGAGGACAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAgGTCACCATGACCTGCAGTGCCAGCTCAAGTGTAAGTTACATGCACTGGTACCAGCAGAAGTCAGGCACCTCCCCCAAAASATGGATTTATGACACATCCAAACTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGTATGGAGGCTGAAGATGCTGCCACTTATTTCTGCCACCAGTGGAGAAgTAACCCATACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCTTCAACAGGGGAG AGTGTTAGTGA

FIG. 11 and FIG. 12 show preferred antibody sequences which have beenannotated to show the CDRs and other feafures.

Guidance regarding humanisation may be found for example in theliterature as published by Greg Winter et al., and techniques for themanipulation and production of recombinant antibodies may be found inHarlow and Lane ‘Antibodies-A Laboratory Manual’, Cold Spring Harbourpress.

In one embodiment, the antibodies (or fragments) may advantageously behumanised by manufacture of chimaeric antibodies.

In another embodiment, the antibodies (or fragments) may advantageouslybe CDR—grafted.

In another embodiment, the antibodies (or fragments) may advantageouslybe fully humanised to the extent that the technology permits.

Binding Affinity

By binding affinity is meant the affinity of binding of the ligand tothe PrP molecule.

As will be appreciated by the skilled reader, high affinity means a lowdissociation constant (Kd). In other words ‘tighter’ binding antibodiessaturate the target sequence at a lower concentration than ‘weaker’binding antibodies. Thus the tighter binding antibodies have a lowerdissociation constant (Kd). Thus an antibody with ‘higher affinity’ incommon parlance is a tighter binding antibody with a lower dissociationconstant (Kd) value.

The ligand needs to have a high enough affinity (i.e. a low enough Kd)for the target sequence of PrP to remain stably bound. For example, ICSM32 (136-143) recognises the target sequence of PrP but did not inhibitbinding to Abeta because it did not remain stably bound to PrP duringthe experiment.

Thus, the affinity of the ligand is suitably 100 nM or less. Moresuitably the affinity of the ligand is 10 nM or less such as 1-10 nM.More suitably the affinity of the ligand is 1 nM or less such as 1 nM to100 pM. Most suitably the affinity is approximately 600 pM, which is theaffinity of ICSM18 (equivalent humanised version is PRN100).

Binding affinity can be measured according to any suitable method knownin the art. For example, affinity values may be determined by followingthe method given in the examples, for example under the heading ‘DELFIA®Assay’ and subheading ‘Antibody Binding (Affinity) Determination’.

Suitably the ligand such as antibody specifically binds to the targetsequence of PrP. A ligand such as antibody “specifically binds” to thetarget sequence of PrP if reacts at a detectable level with the targetsequence of PrP, and does not react detectably with peptides containingan unrelated or different sequence. Binding properties may be assessedas described.

Administration/Formulation

Methods of administering the ligands that target prion proteins forinhibition of oligomeric A[beta] binding, and for other diseasesinvolving prion proteins in the nervous system, are well known in theart and incorporated herein as methods of administering the ligandsaccording to the invention. Compositions involving the ligands of theinvention are also well-known in the art and incorporated herein byreference for use in preventing or treating medical conditions linked toA[beta] oligomerisation.

The ligand used in the methods of the invention may be formulated intopharmaceutical compositions for administration to mammals, includinghumans. The pharmaceutical compositions, which may be used in themethods of this invention, may comprise one or more pharmaceuticallyacceptable carriers, including, e.g., ion exchangers, alumina, aluminumstearate, lecithin, serum proteins, such as human serum albumin, buffersubstances such as phosphates, glycine, sorbic acid, potassium sorbate,partial glyceride mixtures of saturated vegetable fatty acids, water,salts or electrolytes, such as protamine sulfate, disodium hydrogenphosphate, potassium hydrogen phosphate, sodium chloride, zinc salts,colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone,cellulose-based substances, polyethylene glycol, sodiumcarboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol and/orwool fat. The compositions used in the methods of the present inventionmay be administered by any suitable method, e.g., parenterally, orally,by inhalation spray, topically, rectally, nasally, buccally, vaginallyor via an implanted reservoir. The term “parenteral” as used hereinincludes subcutaneous, intravenous, intramuscular, intra-articular,intra-synovial, intrastemal, intrathecal, intrahepatic, intralesionaland intracranial injection or infusion techniques.

Ligands used in the methods of the invention may act in the nervoussystem to inhibit suppression of long term potentiation, and/or toincrease acute memory retention, and/or to improve spatial memoryperformance. Accordingly, in certain methods of the invention, theligand may be administered in such a way that it crosses the blood-brainbarrier. This crossing can result from the physico-chemical propertiesinherent in the ligand molecule itself, tagging or linking the ligand toa vehicle to facilitate crossing the blood-brain barrier, or from othercomponents in a pharmaceutical formulation, or from the use of amechanical device such as a needle, cannula or surgical instrument tobreach the blood-brain barrier. Where the ligand is a molecule that doesnot inherently cross the blood-brain barrier, e.g. a fusion to a moietythat facilitates the crossing, suitable routes of administration are,e.g., intrathecal or intracranial. Where the ligand is a molecule thatinherently crosses the blood-brain barrier, the route of administrationmay be by one or more of the various routes described below.

Suitably the ligand or composition comprising same may be administeredintracerebrally or more suitably peripherally e.g. by intravenous orsubcutaneous injection.

Sterile injectable forms of the compositions described may be aqueous oroleaginous suspension. These suspensions may be formulated according totechniques known in the art using suitable dispersing or wetting agentsand suspending agents. The sterile, injectable preparation may be asterile, injectable solution or suspension in a non-toxic parenterallyacceptable diluent or solvent, for example as a suspension in1,3-butanediol. Among the acceptable vehicles and solvents that may beemployed are water, Ringer's solution and isotonic sodium chloridesolution, hi addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose, any bland fixed oilmay be employed including synthetic mono- or di-glycerides. Fatty acids,such as oleic acid and its glyceride derivatives are useful in thepreparation of injectables, as are natural pharmaceutically acceptableoils, such as olive oil or castor oil, especially in theirpolyoxyethylated versions. These oil solutions or suspensions may alsocontain a long-chain alcohol diluent or dispersant, such ascarboxymethyl cellulose or similar dispersing agents which are commonlyused in the formulation of pharmaceutically acceptable dosage formsincluding emulsions and suspensions. Other commonly used surfactants,such as Tweens, Spans and other emulsifying agents or bioavailabilityenhancers which are commonly used in the manufacture of pharmaceuticallyacceptable solid, liquid, or other dosage forms may also be used for thepurposes of formulation. Compositions containing the ligand according tothe invention may contain suitable pharmaceutically acceptable carriers.For example, they may contain excipients and/or auxiliaries thatfacilitate processing of the active compounds into preparations designedfor delivery to the site of action. Suitable formulations for parenteraladministration include aqueous solutions of the active compounds inwater-soluble form, for example, water-soluble salts. In addition,suspensions of the active compounds as appropriate oily injectionsuspensions may be administered. Suitable lipophilic solvents orvehicles include fatty oils, for example, sesame oil, or synthetic fattyacid esters, for example, ethyl oleate or triglycerides. Aqueousinjection suspensions may contain substances that increase the viscosityof the suspension include, for example, sodium carboxymethyl cellulose,sorbitol and dextran. Optionally, the suspension may also containstabilizers. Liposomes also can be used to encapsulate the molecules ofthe invention for delivery into cells or interstitial spaces. Exemplarypharmaceutically acceptable carriers are physiologically compatiblesolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, water, saline,phosphate buffered saline, dextrose, glycerol, ethanol and the like, insome embodiments, the composition comprises isotonic agents, forexample, sugars, polyalcohols such as mannitol, sorbitol, or sodiumchloride. In some embodiments, the compositions comprisepharmaceutically acceptable substances such as wetting or minor amountsof auxiliary substances such as wetting or emulsifying agents,preservatives or buffers, which enhance the shelf life or effectivenessof the active ingredients.

Parenteral formulations may be a single bolus dose, an infusion or aloading bolus dose followed with a maintenance dose. These compositionsmay be administered at specific fixed or variable intervals, e.g., oncea day, or on an “as needed” basis.

Certain pharmaceutical compositions used in the methods of thisinvention may be orally administered in an acceptable dosage formincluding, e.g., capsules, tablets, aqueous suspensions or solutions.Certain pharmaceutical compositions also may be administered by nasalaerosol or inhalation. Such compositions may be prepared as solutions insaline, employing benzyl alcohol or other suitable preservatives,absorption promoters to enhance bioavailability, and/or otherconventional solubilizing or dispersing agents. Compositions of theinvention may be in a variety of forms, including, for example, liquid(e.g., injectable and infusible solutions), dispersions, suspensions,semisolid and solid dosage forms. The preferred form depends on the modeof administration and therapeutic application. For treating tissues inthe central nervous system, administration can be, e.g., by injection orinfusion into the cerebrospinal fluid (CSF). Administration can also bewith one or more agents capable of promoting penetration of a ligandacross the blood-brain barrier.

The composition can be formulated as a solution, microemulsion,dispersion, liposome, or other ordered structure suitable to high drugconcentration. Sterile injectable solutions can be prepared byincorporating the active ingredient in the required amount in anappropriate solvent with one or a combination of ingredients enumeratedabove, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the active ingredient into asterile vehicle that contains a basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-dryingthat yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution. Theproper fluidity of a solution can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prolonged absorption of injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

The ligand according to fhe invention can be formulated with acontrolled-release formulation or device. Examples of such formulationsand devices include implants, transdermal patches, and microencapsulateddelivery systems. Biodegradable, biocompatible polymers can be used, forexample, ethylene vinyl acetate, polyanhydrides, polyglycolic acid,collagen, polyorthoesters, and polylactic acid. Methods for thepreparation of such formulations and devices are known in the art. Seee.g., Sustained and Controlled Release Drug Delivery Systems, J. R.Robinson, ed., Marcel Dekker, Inc., New York, 1978.

Injectable depot formulations can be made by forming microencapsulatedmatrices of the drug in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the polymer employed, the rate of drug release can becontrolled. Other exemplary biodegradable polymers are polyorthoestersand polyanhydrides. Depot injectable formulations also can be preparedby entrapping the drug in liposomes or microemulsions.

Supplementary active compounds can be incorporated into the compositionsused in the methods of the invention. These can be therapeutic agentseffective to treat, ameliorate or prevent Alzheimer's disease, such asan adrenergic, anti-adrenergic, anti-androgen, antianginal,anti-anxiety, anticonvulsant, antidepressant, anti-epileptic,antihyperlipidemic, antihyperlipoproteinemic, antihypertensive,anti-inflammatory, antiobessional, antiparkinsonian, antipsychotic,adrenocortical steroid; adrenocortical suppressant; aldosteroneantagonist; amino acid; anabolic steroid; analeptic; androgen; bloodglucose regulator; cardioprotectant; cardiovascular; cholinergic agonistor antagonist; cholinesterase deactivator or inhibitor, such asgalantamine, rivastigmine, tacrine and donepezil; cognition adjuvant orenhancer; dopaminergic; enzyme inhibitor, estrogen, free oxygen radicalscavenger; GABA agonist; glutamate antagonist; hormone;hypocholesterolemic; hypolipidemic; hypotensive; immunizing;immunostimulant; monoamine oxidase inhibitor, neuroprotective; N-methylD-aspartate (NMDA) antagonist, such as memantine; AMPA antagonist,competitive or -non-competitive NMDA antagonist; opioid antagonist;potassium channel opener; non-hormonal sterol derivative; post-strokeand post-head trauma treatment; prostaglandin; psychotropic; relaxant;sedative; sedative-hypnotic; selective adenosine antagonist; serotoninantagonist; serotonin inhibitor; selective serotonin uptake inhibitor;serotonin receptor antagonist; sodium and calcium channel blocker;steroid; stimulant; and thyroid hormone and inhibitor agents. The amountof ligand that may be combined with the carrier materials to produce asingle dosage form will vary depending upon the host treated, the typeof antagonist used and the particular mode of administration. Thecomposition may be administered as a single dose, multiple doses or overan established period of time in an infusion.

Dosage

Dosage regimens also may be adjusted to provide the optimum desiredresponse (e.g., a therapeutic or prophylactic response).

Typically, a physician will determine the actual dosage which will bemost suitable for an individual subject. The specific dose level andfrequency of dosage for any particular patient may be varied and willdepend upon a variety of factors including the activity of the specificcompound employed, the metabolic stability and length of action of thatcompound, the age, body weight, general health, sex, diet, mode and timeof administration, rate of excretion, drug combination, the severity ofthe particular condition, and the individual undergoing therapy.

Typically if injected peripherally dosage would be higher, and ifinjected into the brain dosage would be lower.

Compositions for administration according to the methods of theinvention can be formulated so that a dosage of 0.001-10 mg/kg bodyweight per day of the ligand is administered. In some embodiments of theinvention, the dosage may be 0.01-1.0 mg/kg body weight per day. In someembodiments, the dosage may be 0.001-0.5 mg/kg body weight per day.

For treatment with an antibody binding the target PrP c sequence, thedosage can range, e.g., from about 0.0001 to 100 mg/kg, and more usually0.01 to 5 mg/kg (e.g., 0.02 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1mg/kg, 2 mg/kg, etc.), of the host body weight. For example dosages canbe 1 mg/kg body weight or 10 mg/kg body weight or within the range of1-10 mg/kg, preferably at least 1 mg/kg. Doses intermediate in the aboveranges are also intended to be within the scope of the invention.Subjects can be administered such doses daily, on alternative days,weekly or according to any other schedule determined by empiricalanalysis. An exemplary treatment entails administration in multipledosages over a prolonged period, for example, of at least six months.Additional exemplary treatment regimes entail administration once perevery two weeks or once a month or once every 3 to 6 months. Exemplarydosage schedules include 1-10 mg/kg or 15 mg/kg on consecutive days, 30mg/kg on alternate days or 60 mg/kg weekly. In some methods, two or moremonoclonal antibodies with different binding specificities areadministered simultaneously, in which case the dosage of each antibodyadministered falls within the ranges indicated.

Doses discussed are for human subjects unless otherwise indicated. Dosesfor other species are adjusted accordingly. In mice, typical doses are 2mg/mouse. This is approximately equivalent to a dose of 30 mg/kg.Suitably dosage is 0.1-30 mg/kg.

Most suitably, each protein may be administered at a dose of from 0.01to 30 mg/kg body weight, preferably from 0.1 to 10 mg/kg, morepreferably from 0.1 to 1 mg/kg body weight.

When administering nucleic acid(s) for protein expression, suitablythose are administered in amounts capable of supporting levels ofexpression corresponding to the administration levels mentioned forproteins. Polynucleotides of the invention may be administered directlyas naked nucleic acid. When the polynucleotides/vectors are administeredas naked nucleic acid, the amount of nucleic acid administered maytypically be in the range of from 1 μg to 10 mg, preferably from 100 μgto 1 mg.

The ligand according to the invention may be used as part of apharmaceutical kit, which may further comprise an administration means.Means for administration include, but are not limited to syringes andneedles, catheters, biolistic injectors, particle accelerators, i.e.,“gene guns,” pneumatic “needleless” injectors, gelfoam sponge depots,other commercially available depot materials, e.g., hydrogels, osmoticpumps, and decanting, polynucleotide coated sutures, skin patches, ortopical applications during surgery.

Each of the pharmaceutical kits can further comprise an instructionsheet for administration of the composition to a mammal. If the ligandis provided in lyophilized form, the dried powder or cake may alsoinclude any salts, auxiliary agents, transfection facilitating agents,and additives of the composition in dried form. Such a kit may furthercomprise a container with an exact amount of sterile pyrogen-free water,for precise reconstitution of the lyophilized components of thecomposition.

The container in which the composition is packaged prior to use cancomprise a hermetically sealed container enclosing an amount of thelyophilized formulation or a solution containing the formulationsuitable for a pharmaceutically effective dose thereof, or multiples ofan effective dose.

The composition is packaged in a sterile container, and the hermeticallysealed container is designed to preserve sterility of the pharmaceuticalformulation until use. Optionally, the container can be associated withadministration means and/or instruction for use.

It has been surprisingly found that prevention of or decrease in PrP:PrPinteraction prevents binding of oligomeric A[beta] to PrP. Preferablythe ligand that prevents or decreases PrP:PrP interactions prevents PrPoligomerisation from monomers, which as a consequence thereof preventsA[beta] binding. This effect would be complementary to direct inhibitionof the ADDL binding site, and/or the target site of PrP describedherein. In such a situation, it would therefore be advantageous toadminister/use a ligand which inhibits PrP:PrP interaction together withligand(s) which bind to the PrP target site described herein.

It may also be advantageous to administer/use a ligand which inhibitsthe ADDL binding region of PrP together with ligand(s) which bind to thePrP target site described herein.

It may also be advantageous to administer a three-way combination ofligands which inhibit PrP:PrP interaction, which inhibit the ADDLbinding region of PrP and ligand(s) which bind to the PrP target sitedescribed herein.

Ligands that can prevent or decrease PrP:PrP interactions are well-knownin the art for treatment of other diseases involving prion proteins,particularly in the central nervous system. Ligands known for their usein preventing or decreasing PrP:PrP interactions, particularly in thecentral nervous system, are incorporated herein by reference as ligandswhich may be used in combination with the ligands of the invention.

Ligands which directly target the ADDL binding site of PrP 95-105 arealso well known as acknowledged above, and are incorporated herein byreference.

Further Components

The method(s) and application(s) of the invention may further compriseadministering an additional therapeutic agent; the invention may relateto compositions such as pharmaceutical compositions comprising a ligandof the invention and an additional therapeutic agent. Additionaltherapeutic agents include, but are not limited to an adrenergic agent,anti-adrenergic agent, anti-androgen agent, antianginal agent,anti-anxiety agent, anticonvulsant agent, antidepressant agent,anti-epileptic agent, antihyperlipidemic agent, antihyperlipoproteinemicagent, antihypertensive agent, anti-inflammatory agent, antiobessionalagent, antiparkinsonian agent, antipsychotic agent, adrenocorticalsteroid agent; adrenocortical suppressant agent; aldosterone antagonistagent; amino acid agent; anabolic steroid; analeptic agent; androgenagent; blood glucose regulator; cardioprotectant agent; cardiovascularagent; cholinergic agonist or antagonist; cholinesterase deactivator orinhibitor, such as galantamine, rivastigmine, tacrine and donepezil;cognition adjuvant or enhancer; dopaminergic agent; enzyme inhibitor,estrogen, free oxygen radical scavenger; GABA agonist; glutamateantagonist; hormone; hypocholesterolemic agent; hypolipidemic agent;hypotensive agent; immunizing agent; immunostimulant agent; monoamineoxidase inhibitor, neuroprotective agent; N-methyl D-aspartate (NMDA)antagonist, such as memantine; AMPA antagonist, competitive or-non-competitive NMDA antagonist; opioid antagonist; potassium channelopener; non-hormonal sterol derivative; post-stroke and post-head traumatreatment; prostaglandin; psychotropic agent; relaxant; sedative;sedative-hypnotic agent; selective adenosine antagonist; serotoninantagonist; serotonin inhibitor; selective serotonin uptake inhibitor;serotonin receptor antagonist; sodium and calcium channel blocker;steroid; stimulant; and thyroid hormone and inhibitor agents.

Further Applications

In principle the invention may be applied to any scenario in which it isdesired to suppress or interfere with the PrP-Abeta interaction itself.In more detail, the invention may in addition be applied to one or moreof inhibiting suppression of long term potentiation, increasing acutememory retention, improving spatial memory performance and/or blockingdisruption of synaptic plasticity.

Also described is a ligand which at least part thereof binds to at leastpart of helix 1 of PrP for use in preventing or improving medicalconditions linked to A[beta] oligomerisation. Suitably the A[beta]oligomer is made from soluble non-fibrillar forms of A[beta]. Suitablythe A[beta] oligomerisation is A[beta] plaque formation. Suitably themedical condition is synaptic plasticity. Suitably the synapticplasticity is manifested as Alzheimer's Disease. Suitably the PrP isPrP. Suitably the ligand is an antibody. Suitably the antibody ishumanised.

Advantages

This binding of aa131-153 of the prion protein imparts severaladvantages over the ligands known in the art which target the ADDLbinding region of to the prion protein. The ADDL binding region has beenlinked with toxicity, for example with toxic signalling. Ligands bindingthis region may contribute to toxic signalling e.g. D13 and IgG P(Solforosi, 2004) or 4H11 (Lefebvre-Roque, 2007). It is an advantage ofthe invention that such toxicity effects may be avoided.

The ligands according to the present invention selectively recognise thephysiological form of PrP, PrP^(C), whereas ligands binding the ADDLbinding region 95-105 also bind to aberrant forms of the prion protein.Therefore, the ligands according to the present invention may helpstabilise the normal form of the protein and in particular do notcontribute to stabilising the aberrant form of the protein which may bethe case for prior art ligands directed to 95-105.

The ADDL binding region has been suggested to be involved in severalpossible physiological functions of PrP ranging from metal orglycosaminoglycan binding, endocytosis or preventing oxidative stress.Therefore, blocking said region such as in the art using ligands binding95-105 may interfere with those physiological functions. It is anadvantage of using the ligands described herein that such problems areavoided.

The fact that the ligand which binds to 131-153 is effective in blockingADDL binding is surprising because the helix 1 region is on thegeometrically opposite side of the prion protein from the ADDL′ bindingsite region (Zahn, 2000) and therefore would in fact be expected to beone of the least active regions given its structural distance from theADDL binding region. This is even more surprising in light of recentstudies that focus on finding possible ligands for other regions of theprion protein^(4,14).

The invention is now described by way of example. These examples areintended to be illustrative, and are not intended to limit the appendedclaims.

EXAMPLES Materials

A[beta]₁₋₄₂ was from California Peptide Research Inc. (Napa, Calif.) andbiotinylated A[beta]₁₋₄₂ with biotin attached to Asp1 using a 6-carbonlinker (bA[beta]₁₋₄₂) was synthesised, and purified by Dr. James I.Elliott at Yale University (New Haven Conn.). Peptide masses andpurities were determined by electrospray ionisation/ion trap massspectrometry and reverse-phase HPLC, respectively. Other reagents werepurchased from Sigma Aldrich unless otherwise stated.

Production of ADDL/bADDL Preparations.

bA[beta]₁₋₄₂ or A[beta]₁₋₄₂ (˜1.25 mg) was weighed into a screw-cap 1.7ml eppendorf tube, dissolved in ice-cold hexafluoro-2-propanol (HFIP) toa concentration of 1 mM, sonicated for 10 min, the tube sealed and leftto stand at room temperature for 1 h. The solution was then transferredto a 2 ml glass vial and the HFIP evaporated under a stream of dryair/N₂ to produce a clear film. The peptide film was dissolved inanhydrous DMSO with vigorous vortexing for 10 min to produce a 5 mMsolution and then diluted to 100 microM in phenol red-free Ham's F12medium (Promocell GmbH, Heidelberg, Germany) and vortexed for 15 sec.Equal volumes of sample were then transferred to two separate sealedglass vials and incubated at room temperature for 16 h. MonomericA[beta]₁₋₄₂ was produced by dissolving A[beta]₁₋₄₂ peptide to 100 microMin 10 mM NaOH (pH 11) for 1 hour and monomeric status was confirmed bysize-exclusion chromatography. Finally, samples were centrifuged at14,200×g for 15 min to remove any large aggregates and the upper 90% foreach solution collected, used immediately, or snap frozen in liquid N₂and stored at −80° C.

Size Exclusion Chromatography.

0.5 ml aliquots of freshly prepared 100 microM A[beta]₁₋₄₂ (10 mM NaOH,pH 11), ADDLs and bADDLs (phenol red-free Ham's F12 medium, 2% DMSO)were injected onto a Superdex 75 10/30 column (GE Healthcare) and elutedwith PBS at a flow rate of 0.8 ml/min using an AKTA FPLC and peptideelution monitored by absorbance at 280 nm.

Sedimentation Velocity Analytical Ultracentrifugation.

Experiments were performed on a Beckman XLI analytical ultracentrifuge.Freshly prepared 100 microM bADDLs (phenol red-free Ham's F12 medium, 2%DMSO) were centrifuged at 50,000 rpm at 20° C. with absorbance datacollected at 278 nm. Sedimentation velocity data were analysed asdescribed^(32,33) and graphically presented in the standard format withthe sedimentation coefficient plotted against the sedimentationcoefficient distribution.

Electron Microscopy.

For analysis a 4 [micro]l drop of bADDLs/ADDLs was loaded ontonegatively glow discharged copper grids which had been previously coatedwith a continuous carbon film. The sample was left to adhere for 30 secand excess solution carefully blotted using grade 4 Whatman paper, thesample stained with of 2% uranyl acetate (6 [micro]l) for 30 sec,blotted and the grid left to air dry. Images were recorded on film at amagnification of 42986× using an FEI Tecnai T12 electron microscopeoperating at 120 kV. Imaging was done at a defocus range of 700 nm to 1microm and an electron dose of 10-20 electrons per Å². Films weredigitised with a step size of 7 microm using a Zeiss SCAI film scanner,giving a pixel size of 1.63 Å.

SDS-PAGE.

Aliquots (5 & 10 [micro]l) of 10 microM bADDL and ADDL preparations wereboiled in sample buffer and electrophoresed on 16% polyacrylamidetris-tricine gels and analysed for Aβ content by comparison to known Aβstandards following visualisation by silver staining. Alternatively, theADDLs/bADDLS were diluted 1:10 (1 microM) in sample buffer and usedwithout boiling for Western blotting with the N-terminal anti-Aβantibody, 6E10 (Signef, Dedham, Mass.). Immunoreactive bands weredetected and quantified using a Licor Odyssey imaging system (LicorBiosciences, Lincoln, Nebr., USA).

Preparation of Human AD Brain Samples.

Three brains were used for this study; one from a 78 year old women witha history of dementia and confirmed Alzheimer's disease pathology (fromAsterand, Detroit, Mich.), a second from an 80 year old female (UCLInstitute of Neurology brain bank) with clinical and pathologicaldiagnoses of AD and the other from a cognitively intact 68 year oldfemale (UCL Institute of Neurology brain bank). Samples of frozenposterior temporal cortex were thawed on ice and gray matter dissectedfor use, chopped into small pieces with a razor blade and thenhomogenised in 5 volumes of ice-cold 20 mM Tris-HCl, pH 7.4, containing150 mM NaCl (TBS) with 25 strokes of a Dounce homogeniser (Fisher,Ottawa, Canada). To isolate water-soluble Aβ, free from membrane-boundor plaque-associated material, homogenates were centrifuged at 91,000 gand 4° C. in a TLA 55 rotor (Beckman Coultour, Fullerton, Calif.) for 78min and the supernatant removed and used. In order to eliminate lowmolecular weight bioactive molecules and drugs, homogenates weredialysed at 4° C. using slide-a-lyzer dialysis cassettes with 2 kDamolecular weight cut-offs (Fisher, Dublin, Republic of Ireland) againsta total volume of 5 l of TBS (with 2 changes) over a 48 h period. Thedialysate was then aliquoted into 1 ml lots and either stored at −80° C.pending use or used directly to measure the amount and form of Aβpresent. For the latter, 0.8 ml of dialysate was immunoprecipitated withAW7³⁴ at a dilution of 1:80 and analysed by western blotting using acombination of the C-terminal monoclonal antibodies, 2G3 and 21F12 eachat a concentration of 2[micro]g/ml (Dr. Peter Seubert, ElanPharmaceuticals, San Francisco, Calif.). Immunoreactive bands werevisualised using a fluorochrome-coupled secondary antibody (Rockland,Gilbertsville, Pa.) and quantified by comparison to synthetic A[beta]standards using a Licor Odyssey imaging system (Licor Biosciences,Lincoln, Nebr., USA).

Generation of FVB/N Congenic PrP Knockout Line.

The FVB/N-Pmp^(o/o) (PrP null) congenic line was generated by 10generations of backcrossing ZH1 PrP null mice³⁵ to FVB/N followed bygenetic testing by Charles River (Margate, UK) using 84 FVB-specific PCRmicrosatellite makers covering 19 chromosomes at approximately 20 cMintervals to select breeding pairs positive for 100% of the FVB-specificmarkers. The selected congenic pairs were inter-bred to remove theendogenous murine PrP gene and to restore homozygosity of the knockoutallele.

In Vitro Electrophysiology.

Male, two to four month old FVB/N (Harlan, Wyton, UK) or PrP null mice(MRC Prion Unit) were used to study the effects of bADDLs andA[beta]-containing extracts of human brain. In addition, two to threemonth old C57BL/6J mice (Charles River, Margate, UK) were used toexamine the effects of bADDLs/ADDLs and the anti-PrP antibodies, ICSM-18and ICSM-35. In all cases, mice were anaesthetised with isoflurane/02and decapitated. The brain was rapidly removed and immersed in ice-coldsucrose-based artificial cerebrospinal fluid (sACSF) containing 87 mMNaCl, 2.5 mM KCl, 7 mM MgSO₄, 0.5 mM CaCl₂, 25 mM NaHCO₃, 25 mM Glucose,1.25 mM NaH₂PO₄ and 75 mM Sucrose. Parasagital sections (350 microm)were prepared on a Leica VT1000S vibratome using stainless steel razorblades (Campden, Loughborough, UK). Slices were immediately transferredto a holding chamber (BSC—PC, Warner Instruments, Hamden, Conn.)containing ACSF: 119 mM NaCl, 2.5 mM KCl, 1.3 mM MgSO₄, 2.5 mM CaCl₂,26.2 mM NaHCO₃, 11 mM Glucose and 1.25 mM NaH₂PO₄. Circulating ACSF wascontinuously bubbled with a mixture of 95% O₂ and 5% CO₂ and slicesallowed to recover for at least 90 min at room temperature.

Extracellular recordings were performed as described previously(O'Nuallain et al. 2010). Briefly, slices were submerged in a recordingchamber and perfused with oxygenated ACSF at a rate of 2-3 ml/min andthe perfusate warmed to 30° C. using an inline heating tube (HPT-2A, ALAScientific Instruments, Westbury, N.Y.). A stainless steelmicroelectrode (FHC, Bowdoin, USA) was used to stimulate Schaffercollateral fibres, and extracellular field EPSPs (fEPSPs) were recordedfrom stratum radiatum of CA1 using a glass microelectrode. fEPSPs wererecorded using a Multiclamp 700B amplifier in tandem with a Digidata1440A digitiser (Axon Intruments). Data were collected using pClamp 10software and analysed using Clampfit 10.2 (Molecular Devices). For allexperiments, test stimuli were given once every 30 sec (0.033 Hz), andthe stimulus intensity was set to give a baseline fEPSP of 40-50% of themaximal response. A stable baseline was recorded for at least 20 minprior to application of ADDLs/antibody. In experiments usingbADDLs/ADDLs (stock solutions were diluted 1:200 into ACSF to producenominal concentrations of 500 nM based on the starting weight ofA[beta]₁₋₄₂ monomer) or TBS extracts of human brain (1 ml of extractdiluted into 20 ml ACSF) the sample was added to the perfusate 30 minprior to induction of LTP. Where a combination of ADDLs and anti-PrPantibodies were used, the antibody was added to the perfusate 20 minprior to the ADDLS. LTP was induced by theta burst stimulation (TB, 10bursts of 4 stimuli at 100 Hz, with an interburst interval of 200 msec)given at baseline intensity. The ACSF was recycled using peristalticpumps (101U/R, Watson-Marlow, UK) ensuring that the ADDLs, brain samplesand/or antibodies were present for the duration of the experiment. LTPis expressed as the mean±SEM % of baseline fEPSP slope. Statisticalcomparisons used ANOVA with post hoc Tukey-Kramer test. All experimentswere interleaved with respect to genotype. In addition, vehicle andADDL/bADDL/human brain derived A[beta] experiments were performed on thesame day ensuring each animal was its own control while alternatingtreatments daily to avoid any temporal bias.

In Vivo Electrophysiology.

In vivo studies on urethane (1.5 gm/kg i.p.) anaesthetised male AdultWistar rats (250-300 g) were approved by Trinity College Dublin'sethical review committee and by the Department of Health, Republic ofIreland. Electrodes were made and implanted as described previously³⁶.Briefly, twisted-wire bipolar electrodes were constructed fromTeflon-coated tungsten wires (62.5[micro]_(m) inner core diameter,75[micro]m external diameter). Single pathway recordings of fEPSPs weremade from the stratum radiatum in the CA1 area of the right hippocampalhemisphere in response to stimulation of the ipsilateral Schaffercollateral—commissural pathway. Electrode implantation sites wereidentified using stereotaxic coordinates relative to bregma, with therecording site located 3.4 mm posterior to bregma and 2.5 mm right ofmidline, and the stimulating electrode located 4.2 mm posterior tobregma and 3.8 right of midline. The optimal depth of the wireelectrodes in the stratum radiatum of the CA1 region of the dorsalhippocampus was determined using electrophysiological criteria andverified post-mortem. Test fEPSPs were evoked at a frequency of 0.033 Hzand at a stimulation intensity adjusted to elicit a fEPSP amplitude of50% of maximum. The high frequency stimulation (HFS) protocol forinducing LTP consisted of 10 bursts of 20 stimuli with an inter-stimulusinterval of 5 msec (200 Hz), and an inter-burst interval of 2 sec. Theintensity was increased to give an EPSP of 75% of maximum amplitudeduring the HFS. To inject samples, a stainless-steel guide cannula (22gauge, 0.7 mm outer diameter, 13 mm length) was implanted above theright lateral ventricle (1 mm lateral to the midline and 4 mm below thesurface of the dura) just prior to electrode implantation. Animalsreceived two intracerebroventricular (i.c.v.) injections via an internalcannula (28 gauge, 0.36 mm outer diameter). The first injectioncontained water vehicle, ICSM-18 or mouse IgG1 isotype control antibody(MAB002, R&D Systems, Minneapolis) in a 10[micro]l volume. The secondinjection (5[micro]l), which contained either water vehicle or humanbrain TBS extract, was administered 30 min later, 15 min before the HFS.The experimenter was blinded regarding treatment group in the experimentdirectly comparing ICSM-18 and control antibody. Verification of theplacement of the cannula was performed post-mortem by checking thespread of i.c.v. injected ink dye. LTP is expressed as the mean±SEM %baseline field EPSP amplitude recorded over at least a 30 minutebaseline period. Similar results were obtained when the EPSP slope wasmeasured. Statistical comparisons used ANOVA with post hoc Tukey test,paired and unpaired Student t-tests or Mann Whitney U-test, asappropriate.

Protein Expression and Purification.

Constructs of human PrP were to expressed³⁷ and purified³⁸ as describedpreviously. Protein quality was confirmed by SDS-PAGE, MALI-TOF massspectrometry and Circular Dichroism spectroscopy.

DELFIA® Assay³⁹.

100[micro]l of 1 [micro]M human PrP (10 mM sodium carbonate, pH 9.6) wasbound to medium binding 96-well white plates (Greiner) overnight at 4°C., washed with 3×300 [micro]l of PBS (0.05% Tween-20), blocked with300[micro]l 2% BSA in PBS (0.05% Tween-20) at 37° C. for 2 h and washedwith 3×300[micro]l of PBS (0.05% Tween-20). If required, 100[micro]l ofantibody was then incubated in PBS (0.05% Tween-20) for 1 hour andwashed with 3×300[micro]l of PBS (0.05% Tween-20). 100[micro]l ofdifferent preparations of A[beta]₁₋₄₂ were incubated in PBS (0.05%Tween-20, 0.1% BSA) for 1 hour and washed with 3×300[micro]l of PBS(0.1% Tween-20). A[beta] was detected by 100[micro]l of 1 [micro]g/ml6E10 in PBS (0.05% Tween-20) for 1 hour, washed with 3×300[micro]l ofPBS (0.05% Tween-20), incubated for 30 min with 300 ng/ml of DELFIA®Eu-N1 anti-mouse antibody in DELFIA® assay buffer, washed with3×300[micro]l of PBS (0.05% Tween-20) before enhancing with 100[micro]lof DELFIA® Enhancement Solution. Biotinylated A[beta] was detected by100[micro]l of 50 pM DELFIA® Eu-N1 Streptavidin (DELFIA® assay buffer)and washed with 3×300[micro]l of PBS (0.05% Tween-20) before enhancingwith 100 [micro]l of DELFIA® Enhancement Solution.

Antibody Binding (Affinity) Determination

For PrP antibody-binding experiments (e.g. determining the affinity ofligands for PrP) ICSM antibodies were incubated for 30 min with 300ng/ml of DELFIA® Eu-N1 anti-mouse antibody in DELFIA® assay buffer,washed with 3×300[micro]l of PBS (0.05% Tween-20) before enhancing with100[micro]l of DELFIA® Enhancement Solution. Binding of antibodies toPrP was detected by incubated for 30 min with 300 ng/ml of DELFIA® Eu-N1anti-mouse antibody in DELFIA® assay buffer, washed with 3×300[micro]lof PBS (0.05% Tween-20) before enhancing with 100 [micro]l of DELFIA®Enhancement Solution. Plates were scanned for time-resolved fluorescenceintensity of the europium probe (λ_(ex)=320 nm, λ_(em)=615 nm) using aPerkin Elmer EnVision plate reader. Binding Constants were calculatedusing a 1-site Langmuir isotherm and were tested for tight bindingcharacteristics using the equationy=(F_(min)+(((L+E_(o)+K_(d))−(((L+E_(o)+K_(d))²)−4*E_(o)*L)^(0.5))/(2*E_(o)))*(F_(max)−F_(min)))where F_(min)=minimum fluorescence, F_(max)=maximum fluorescence,E_(o)=the total concentration of binding sites, K_(d)=the dissociationconstant and L=total A[beta] concentration. This ensures a true K_(d)can be measured and that direct saturation of the protein surface assoon as stoichiometric quantities of peptide are added has not occurred.

Molecular Modelling.

A model of the full ISCM-18 antibody complex with human PrP^(C) ₁₁₉₋₂₃₁was constructed as follows. Two copies of the corresponding PrP-Fabcomplex (2W9E) were superimposed onto on the Fab domains of a full humanIgG antibody structure (1 HZH). The human Fab domains were removed andthe loops between the 1 HZH-Fc and 2W9E-Fab domains rebuilt.

Example 1

The ADDL particle was constructed by first building a full atomic modelof Aβ₁₋₄₂ as an extended β strand with uncharged side-chains andtermini. This molecule was subjected to molecular dynamics simulation at1000K for 0.1 nsec and structures saved every 1 psec. The chaincollapsed into a globular structure after 25 psec of simulation and the57 most compact structures were chosen from the last 75 psec of thesimulation. The maximum spatial extent of these selected molecules wasmeasured (in all three dimensions) and found to be just below 3.0 nm.These 57 molecules were placed at random in the 57 vacant sites ofspherical array made by removing the appropriate 8×8.5 corner sites(i.e. 7 per corner and 3 shared with adjacent corners) of a 5×5×5 cubicarray with a 3.0 nm lattice spacing. The protonation state of the57Aβ₁₋₄₂ molecules was reset to correspond to pH 7, water molecules wereadded in a 3.5 Å layer around each polypeptide and the complex relaxed2000 steps of energy minimisation. The complex was subjected to 0.1 nsecof molecular dynamics at 300K resulting in a roughly spherical, hydratedADDL model with a diameter around 10 nm (and a corresponding proteindensity of 0.82 gl⁻¹).

The N-terminus of the PrP c molecule, taken from the Fab crystalstructure complex (2W9E), was extended back to residue 95 as anunstructured polypeptide. Copies of this molecule were manually dockedto the surface of the ADDL particle in an annulus around the “equator”to illustrate how lateral interactions between such surface-bound prionmolecules might occlude helix 1 and so compromise ISCM-18 binding.Molecular graphics and model building was performed using InsightII(2005) and energy calculations using Discover 2.98 (Accelrys).

Example 1 Characterisation of Reproducible Preparations of ADDLs

A standardised procedure to generate ADDL preparations from A[beta]₁₋₄₂or bADDL preparations from biotinylated A[beta]₁₋₄₂ (bA[beta]₁₋₄₂) wasused and then assessed the consistency of the species produced using abattery of biophysical tests. Both normal and biotinylated A[beta] wereused to confirm that biotinylation did not fundamentally alter theproperties of the oligomers. Size-exclusion chromatography (SEC) using 3different running buffers showed that both ADDLs and bADDLs had highlysimilar profiles producing two prominent peaks (FIG. 1 a and FIG. 2).The first peak eluted in the void volume and the second eluted in avolume consistent for Aβ monomer²¹. Moreover, both peaks had trailingand leading shoulders suggesting the presence of low abundance oligomersintermediate in size between Aβ monomer and the void material. Given thelimited size resolution of SEC, and the possibility of forming artefactsowing to non-ideal interactions with the column, the bADDL preparationswere also analysed using a solution-based technique, analyticalultracentrifugation (FIG. 1 b). Sedimentation of bADDLs occurred in twodistinct phases containing approximately 60 and 40% of the material,respectively. The initial sedimentation contained a mixture of specieswith calculated masses ranging from 90-400,000 and accounted for around60% of the peptide. The slowly sedimenting portion contained a singlespecies with calculated molecular weight of 5-6,000 close to the valueexpected for monomeric, biotinylated A[beta] peptide. In agreement withthe SEC results, the AUC data confirm that bADDL preparations areheterogeneous and include Aβ monomer, small amounts of low n-oligomersand species with calculated masses >90,000. It is noteworthy that in allsamples tested bADDL preparations contained slightly greater amounts ofhigh molecular weight species than did ADDLs (FIGS. 1 a & 2). Consistentwith other biophysical assessments, negative-stain electron microscopyconfirmed that ADDL and bADDL preparations contained a mixture ofdifferent sized species, ranging from globular structures of 8-12 nmdiameter and flexible rods of 15-60 nm in length and 5-10 nm diameter(FIG. 1 c and FIG. 3). These structures are reminiscent of thosedetected by Lauren et al.¹, but are in contrast to the entirely globularstructures observed by Chen et al.¹⁴ when prepared using simplephosphate buffer. The species detected by SEC, AUC and EM wereSDS-labile and migrated on SDS-PAGE predominantly as monomer (FIG. 1 d),however a small amount of higher molecular weight Aβ species weredetected if samples were not boiled prior to SDS-PAGE and proteinsvisualised by Western blotting. Since a major factor which has beenfound to influence production of ADDL preparations of consistentcomposition is the effective solubilisation of HFIP-treated Aβ, SDS-PAGEand silver staining as a simple means to measure the total amount of Aβpresent in all the ADDL preparations was used. These experimentsrevealed that the actual concentration of total Aβ in the ADDLpreparations varied between 70 to 90 microM (based on the molecularweight of Aβ monomer), less than the 100 microM concentration based onthe starting amount of Aβ used.

Example 2 Both AD Brain-Derived A[Beta] and ADDLs Inhibit LTP in aPrP-Dependent Manner

Having established procedures to produce and characterise the bADDL andADDL preparations, their effect on synaptic plasticity and to determineif this effect required expression of PrP was assessed. As expected,both the bADDL and ADDL preparations significantly inhibited LTP inhippocampal slices from wild type FVB/N and C57B6/J mice (P<0.01, FIG. 4a and FIG. 5). Using an FVB/N congenic PrP-null mouse line was nextinvestigated if this Aβ-mediated inhibition of LTP required theexpression of PrP. As in a prior study using another PrP null mousemodel¹, bADDLs that blocked LIP in wild type mice failed to impair LTPin the hippocampi of the PrP null mice (FIG. 4 b). While these resultssuggest a potential role for PrP in bADDL-mediated depression ofhippocampal LTP it is not clear if such preparations include Aβ speciesthat occur in AD brain. Therefore, the fact if water-soluble extracts ofAD brain that contain SDS-stable Aβ dimers also required the expressionof PrP for their plasticity impairing effects was determined. For theseexperiments a brain extract from a non-demented control subject thatlacked detectable Aβ and a tris-buffered saline (TBS) extract from an ADbrain that contained significant amounts of Aβ monomer and SDS-stabledimer (FIG. 4 c) were used. As in earlier experiments²² theAβ-containing extracts potently inhibited LTP in slices from FVB/N mice(FIG. 4 d, P>0.05), whereas TBS extracts that lacked Aβ had no effect(FIG. 4 d). Importantly, as was the case with bADDLs (FIG. 4 b), theblock of LTP mediated by brain-derived Aβ required the expression ofPrP^(C), with the AD-TBS extract unable to alter LTP in hippocampalslices from PrP null mice (FIG. 4 d). As expected, application of TBSfrom a control brain, that lacked detectable amounts of Aβ, had noeffect on LTP in slices from either PrP expressing or PrP null FVB/Nmice (FIG. 4 d and 168±10% vs 164±10%, respectively). These findingsprovide important evidence that PrP is required for the plasticityimpairing effects of pathogenically relevant brain-derived Aβ speciesand suggest that PrP may be required for the changes in synapticfunction that characterise the earliest stages of AD²³. With regard tohow the active A[beta] species in human brain relate to the activespecies present in an Aβ preparation such as ADDLs, it is worth pointingout that both sources are heterogenous. In the case of aqueous extractsof human brain, SDS-stable dimers have been shown to mediatesynaptotoxicity, however, these need not necessarily exist as discreteAβ dimers, but could also include larger assemblies built fromSDS-stable dimers^(22,24) some of which may overlap in size andstructure with assemblies present in the highly heterogenous ADDLpreparation (FIG. 1 and Hepler et al²⁰).

Example 3 High Affinity PrP:ADDL Interaction can be Targeted at MultipleSites

Having established the requirement for PrP for the plasticity impairingactivity of both AD brain-derived Aβ and ADDLs, the binding of activepreparations of Aβ to PrP was investigated. Previous characterisation ofthe PrP:A[beta] interaction has been carried out using preparations ofsynthetic Aβ that acted in a PrP-independent manner³, had not been shownto be toxic^(4,14) or were studied at micromolar concentrations^(3,14).The interaction using a high-throughput plate-based DELFIA® assay wasprobed. A binding response between huPrP₂₃₋₂₃₁) and either bADDLs orADDLs was detected at low nanomolar concentrations with apparentdissociation constants for total A[beta] of 82±7 nM and 100±30 nM,respectively (FIG. 6 a), with no indication of a tight-binding component(see materials and methods). Given that the total concentration of Aβpresent in the stock solution was on average 20% less than the 100microM value derived from the weight of starting peptide powder, that asignificant portion of the peptide remained as monomer (FIGS. 1 a andb), and that the molar concentration of Aβ oligomers must (because oftheir higher molecular weight) be lower than the concentration based onmonomer content, it is evident that one or more of the species presentin ADDL preparations binds PrP very tightly, probably in the picomolarrange. In contrast, Aβ monomer exhibited no binding to PrP atconcentration ≦3×10⁻⁷M (FIG. 13 a). Non-specific oligomer binding to abackground BSA surface was observed for both ADDLs and bADDLs when theconcentration was raised from high nanomolar to low micromolar,highlighting the importance of probing this interaction in the nanomolarrange using well defined oligomer preparations. Moreover, the modestbinding observed when micromolar concentrations of monomer were usedlikely resulted because spontaneous aggregation of Aβ occurs in thisconcentration range²⁴ and once formed such aggregates could bind to PrP.Consequently, all subsequent experiments were carried out using bADDLsat a concentration of 100 nM (monomer equivalent A[beta]) where the mostspecific interaction could be measured. Given that both PrP²⁵ andA[beta]²⁶ are known to contain high affinity copper binding sites inrelevant positions, the possibility that their interaction may bemediated by copper chelation was investigated. Addition of up to 10 mMEDTA did not change the level of bADDL binding (FIG. 7). At theseconcentrations EDTA should be capable of displacing copper from both PrPand A[beta]²⁷ thus excluding simple, non-specific copper chelation asthe mechanism for the PrP:A[beta] interaction. The binding to differentlength constructs of PrP showed that huPrP₉₁₋₂₃₁ bound similarquantities of bADDLs as did the full length construct, suggesting thecrucial high affinity binding site was not in the region 23-91 (FIG. 6b). In contrast, huPrP₁₁₉₋₂₃₁ displayed almost no binding, confirmingthe role of the 91-119 region in the high affinity PrP:A[beta]interaction. A competition assay, whereby increasing concentrations ofthe different PrP constructs were co-incubated with bADDLs to preventbinding to surface-bound huPrP₂₃₋₂₃₁, confirmed that the interactioncould occur in solution and revealed that whilst the region 23-90 didnot appear to contain a separate high-affinity binding site, it wasindeed involved in modulating the high affinity interaction (FIG. 6 c).

The ability of the antibody ICSM-35, which binds to an epitope containedin residues 95-105 of PrP⁷, the putative site of ADDL binding¹ was thentested for its ability to block the PrP:Aβ interaction. This antibodyblocked binding of bADDLs to PrP in a classical dose-dependent mannerwith an IC₅₀=10.4±1.7 nM (FIG. 6 d). A screen of 28 PrP-bindingantibodies pre-incubated with PrP prior to the addition of bADDLs showedthat all antibodies that bound fully to PrP were capable of blockingbADDL binding, at least in vitro, although their efficacy varied (FIG. 6e). The differences in efficacy were clearly epitope dependent, withthose interacting directly with the putative ADDL binding site mosteffective, followed by those that bind to helix 1 of PrP, while thosebinding to other structured regions of the protein were the leasteffective. There was no epitope-dependent correlation between the levelof antibody binding and the level of inhibition although, as expected,individual antibodies that failed to remain bound to PrP did not inhibitbADDL binding. Furthermore, the two best characterised antibodies thatbind to helix 1 and the 95-105 epitope (ICSM-18 and ICSM-35,respectively), both bind to full-length human prion protein withaffinities of approximately 10 nM, block A[beta] oligomer withinhibition constants of approximately 20 nM, yet still differ in themagnitude of their inhibition (FIG. 13) ICSM-18 and ICSM-35 were chosenfor further characterisation as representative members of the groupsthat bind to helix 1 and the 95-105 epitope respectively, due to theirproven efficacy as anti-prion therapeutics without causing acutetoxicity⁶ and because the structure of the PrP:ICSM-18 complex has beensolved at atomic resolution⁸.

The ability of ICSM-18 to block the interaction, despite binding to anepitope far-removed from the 95-105 segment, is surprising (FIG. 6 f).It is unlikely that the on-rate of PrP with the A[beta] is reducedsignificantly by linking with the antibody, since the enlarged structurewill diffuse only slightly more slowly. The dissociation rate of PrPfrom the A[beta] structure is not likely to increase by linkinginteractions with the antibody owing to bulk-solvent effects. Theantibody is also unlikely to cause disruption of the water structure ordielectric properties around the PrP:A[beta] interface; hence,hydrophobic or electrostatic interactions will be largely unaffected.However, if PrP:PrP contact is needed to stabilize the PrP layer adheredto the A[beta] aggregates, then interaction with the antibody wouldcertainly prevent this and weaken the system. Multiple PrP binding siteswould suggest the oligomer contains a repeat structure. It may be thatsuch antibodies prevent the binding of larger Aβ assemblies but notsmaller species. Likewise, reorganisation of the protein on the membranesurface could bring these epitopes into close proximity allowing ICSM-18to sterically block the interaction, although this is unlikely to be thecause of the effect in a plate-based biochemical assay and would notexplain the stronger inhibition of helix 1 directed antibodies comparedto those that bind to structured areas closer to the A[beta] oligomerbinding site. Either option opens up the possibility of using multipleantibodies to therapeutically block this interaction. Moreover, theseresults validate the use of this novel high throughput system as auseful first round screen to identify candidate therapeutics capable ofinhibiting or modulating ADDL binding to PrP.

Example 4 Therapeutic Antibodies Block the Aβ-Mediated Disruption of LTPin Vivo

To further assess the potential of two lead monoclonal antibodiesidentified in our screen, and that belong to the two groups ofantibodies that most effectively blocked ADDL binding to PrP in vitro(FIG. 6 e), it was examined if these antibodies could also blockAβ-mediated impairment of synaptic plasticity. To ensure the effect wasnot just present it FVB/N mice, this part of the study was carried outusing in the C57B6/J model, more commonly used for electrophysiologicalstudies. Perfusion of hippocampal slices from C57B6/J mice with ADDLs 30min prior to LTP induction significantly depressed LTP compared toslices treated with buffer control alone (FIG. 9 a, p<0.01), whereasprior application of low concentrations of the anti-PrP antibody,ICSM-35 (2 [micro]g/ml, 13 nM) abolished the ADDL-mediated impairment ofLTP (FIG. 9 a, p<0.01). Similarly, when slices were incubated withICSM-18 20 min prior to ADDLs this antibody also protected against theADDL-mediated block of LTP (FIG. 9 b, p<0.01). Importantly both ICSM18and ICSM35 had no significant effect on LTP when administered alone(FIG. 8 a & b). ICSM-35 is directed against amino acids 93-102 of PrP cwhich includes the Aβ:PrP binding domain identified in this and priorstudies¹, whereas, ICSM-18, selectively binds to helix 1 of PrP⁸. Thusunlike ICSM-35, which should directly target the ADDL binding site,ICSM-18 may act by hindering formation of the PrP:A[beta] interaction.

Having found that anti-PrP antibodies prevented ADDL-mediated inhibitionof LTP in mouse hippocampal slices, next it was examined the in vivoefficacy of one of the antibodies, ICSM-18, in a different species, therat. This would confirm if the PrP-dependence of A[beta] oligomertoxicity was species as well as mouse strain independent. A comparisonof the ability of ICSM-18 with an IgG1 isotype control antibody foabrogate the inhibition of hippocampal LTP by the pathophysiologicallyrelevant Aβ-containing TBS extract of AD brain was made. In addition, toconfirm that the involvement of PrP was generalisable extracts fromdifferent AD and control brains than those used in FIG. 4 d were used.Intracerebroventricular (i.c.v.) pre-injection of the anti-PrP antibodycompletely prevented the AD brain Aβ-mediated inhibition of highfrequency stimulation (HFS)-induced LIP. In contrast, animals injectedwith AD brain extract immunodepleted of Aβ (FIG. 10) no longer blockedLTP (131±6, n=6; p<0.05 compared with baseline; p>0.05 compared withvehicle injected controls at 3 h). Thus, acute administration of solubleextract from AD brain (5 [micro]l, i.c.v.) (FIGS. 9 c and 10) completelyinhibited LIP at 3 h post-HFS in an Aβ-dependent manner in animalsinjected 30 min previously with the control antibody (30 [micro]_(g) in10 [micro]l, i.c.v.) (p>0.05 compared with baseline; p<0.05 comparedwith controls that received two vehicle injections). In contrast, inanimals that were pre-injected i.c.v. with ICSM-18 (30 [micro]g) HFSinduced robust LIP (p<0.05 compared with baseline and compared with ADbrain Aβ+control IgG1) that was similar in magnitude to controls(p>0.05). When injected alone, neither ICSM 18 nor the control IgG1significantly affected the magnitude of LTP (FIG. 8 c). The finding thatsequence-selective targeting of PrP using antibodies can ameliorate theplasticity impairing activity of AD brain-derived material in rats invivo corroborates the in vitro finding with ADDLs and strongly supportsfurther exploration of this approach as an attractive therapeuticstrategy.

To highlight the importance of the helix-1 epitope as a possibletherapeutic target for abeta toxicity independently of the antibodyscaffold we tested the ability of a humanised (IgG4) form of ICSM18(PRN100) to block AD brain Abeta-mediated inhibition of high frequencystimulation (HFS)-induced LTP. Results are shown in FIG. 14. PRN100completely blocked the inhibition of LTP (grey triangles) seen when ADbrain was injected alone (red circle) or in the presence of a controlhuman IgG4 (pink triangles), compared to vehicle control (blacksquares). This confirms that ligands such as engineered antibodies thatbind to helix-1 could be used to target PrP in the brain and block toxiceffects linked to human Alzheimer's disease. That this has beensuccessfully achieved with a fully humanised antibody furtherdemonstrates their therapeutic potential.

Conclusions from the Examples

These data support the earlier finding that PrP c acts as a receptor formediating toxicity of certain Aβ species. That the inhibitory effect ofADDLs on synaptic plasticity is PrP-dependent has been confirmed usingin vitro LTP recordings from congenic wild type and PrP null mice andimportantly that PrP expression is required for the plasticity-impairingactivity of human brain-derived Aβ. There has been much debate about thenature of biologically relevant A[beta] oligomers. Here two distinctpreparations were used, one prepared from synthetic Aβ₁₋₄₂ to formADDLs, and which were confirmed to be biologically active and the otherderived from the water-soluble phase of human AD brain. By using ADDLswhich are known to be active and to have similar biophysicalcharacteristics as those used by Lauren et. al the veracity of earlierreports that Aβ toxicity was mediated (at least in part) through PrPcould be tested. Importantly, both preparations inhibited LTP in aPrP-dependent manner, suggesting that the ADDL preparation contained acomponent with similar properties to those found in AD brain.Heterogeneous preparations of Aβ aggregates are known to havenon-specific cytotoxicity at high concentrations and it would thereforebe incorrect to interpret a failure of PrP targeting to ameliorate suchnon-specific toxicity as excluding a role for PrP in Aβ mediatedneurotoxicity. AD is a clinicopathological syndrome not a singledisease, with multiple aetiologies. To expect PrP ablation to blocktoxicity in all aspects of all models would be to oversimplify a complexproblem. The dependence of toxicity on particular receptors inindividual animal models of AD may allow us to dscertain which modelscorrectly mimic particular aspects of AD. A number of synaptic proteinshave been shown to affect the binding and toxic effects of A[beta].mGluR5 was shown to affect binding of Aβ oligomers to excitary synapseswith anti-mGluR5 receptors antibodies reducing A[beta] oligomer bindingby 50%²⁸. This is a similar level of reduction shown by PrP¹. Whilst theeffect of this receptor on A[beta] binding was directly visualised, abinary interaction between A[beta] and mGluR5 has not been proven. EphB2was recently shown to co-precipitate with cell derived oligomers andfibronectin repeat domain was shown to be critical²⁹. Again, a directbinary interaction has not been proven. The LIP deficit in the J20 mousemodel of AD, caused by down-regulation of NMDAR, was reversed by overexpressing EphB2 and it would be interesting to see if this also appliesto exogenous human brain-derived A[beta] oligomers or whether thisrequires in situ A[beta] present over longer periods. Previous studieshave shown NMDAR are involved in toxic effects related to ADDLs butappear not to bind directly³⁰. Given that PrP has been suggested tointeract with NR2D subunits and attenuate excitotoxicity³¹ it isplausible that a number of these proteins are involved in A[beta]toxicity through similar pathways. In that sense, the lack of a vitalPrP function would make it a most attractive therapeutic target.

In addition, the demonstration that A[beta]-mediated inhibition of LTPin vivo and in vitro can be blocked by anti-PrP antibodies furtherextends these findings, arguing against the effect in PrP null micebeing due to unknown protective effects of constitutive PrP ablation.That antibodies raised against two structurally and sequentiallydifferent regions of the protein are active strongly argues that PrP isthe target of these antibodies in vivo and that the effect is notnon-specific. Furthermore, these same antibodies have already been usedto successfully treat prion disease in mice without causing toxiceffects⁶. The PrP:Aβ binding interaction has been further characterisedusing material of known biological activity and a biophysical assaydeveloped to investigate potential therapeutic agents which mightefficiently disrupf this interaction. The anti-PrP monoclonal antibodiesICSM-18 and 35, already extensively studied in vivo in mouse and fullyhumanised for investigation as putative human anti-prion therapeutics,potently inhibit Aβ-induced effects on synaptic plasticity both in vitroand in vivo suggesting that these and/or humanised versions of theseantibodies find application as AD therapeutics either individually or incombination. Since both ADDL preparations and Aβ extracted from humanbrain in aqueous buffer are highly heterogeneous, additional studies mayhelp to biophysically characterise the key toxic species that bind toPrP.

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1. A ligand capable of stably binding PrP at a site within amino acidresidues 131 to 153 of PrP, for use in treatment or prevention of atleast one of Alzheimer's Disease, toxicity of Aβ oligomers, and impairedsynaptic plasticity, wherein said ligand is an antibody, say, Fab, orother antigen binding fragment thereof. 2-4. (canceled)
 5. A ligandaccording to claim 1 wherein said ligand binds PrP at a site withinamino acid residues 131 to 153 of PrP with an affinity value 100 nM orless.
 6. A ligand according to claim 1 wherein amino acid residues 131to 153 of PrP have the sequence GSAMSRPIIHFGSDYEDRYYREN.
 7. A medicamentfor treatment of impaired synaptic plasticity, or toxicity of Aβoligomers, or Alzheimer's Disease comprising a ligand as defined inclaim
 1. 8. A medicament for prevention of impaired synaptic plasticity,or toxicity of Aβ oligomers, or Alzheimer's Disease comprising a ligandas defined in claim
 1. 9. A method of treatment of impaired synapticplasticity, or toxicity of Aβ oligomers, or Alzheimer's Disease, saidmethod comprising administering to a subject a therapeutically effectiveamount of a ligand as defined in claim
 1. 10. A method of prevention ofimpaired synaptic plasticity, or toxicity of Aβ oligomers, orAlzheimer's Disease, said method comprising administering to a subject atherapeutically effective amount of a ligand as defined in claim
 1. 11.A ligand according to claim 1 wherein said ligand comprises an antibodyhaving at least the complementarity determining sequences (CDRs) ofFIGS. 11 and
 12. 12. A ligand according to claim 1 wherein said ligandcomprises ICSM18 antibody.